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A new mathematical model for relative quantification in real-time RT-PCR
Abstract
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
... 1.94 means 94% amplification efficiency). Relative expression levels were determined with efficiency correction, which considers differences in primer pair amplification efficiencies between target and reference gene and results in a more reliable estimation of the "relative expression ratio" through the 2ΔCt method [7][8]. Expression data and associated technical errors on duplicates were calculated using the gene expression module of the BIORAD iQ5 software, which follows models and error propagation rules outlined in the geNorm manual. ...
... In this respect, the genes coding for hydrophilins, proteins linked to the stress due to the dehydration of the microorganisms, have been of particular interest. In recent years, they have been the subject of some studies to determine the levels of expression of these genes [1][2][3][4][5][6][7][8][9]. The ability of micro-organisms to adapt rapidly to changing environmental conditions is essential for their survival. ...
... In strain F12-7, the TIF11 and YJL144W genes were the least expressed. The TIF11 and YJL144W genes have been reported to be important in strengthening the ability of yeasts to resist water stress [1][2][3][4][5][6][7][8][9]. Our result was in line with those of Cordero-Otero et al. [9]. ...
... The color code indicates the p-value of a two sided Student's t test of each mutant versus the respective wild type before and after Benjamini-Hochberg correction. Expression levels were calculated with the ΔΔCt method using ACT8 (AT1G49240) as a standard (Pfaffl 2001) and calibrated by biological replicate 1 (rep1 ) of Col-0. ...
... The color code indicates the p-value of a two sided Student's t test with Benjamini-Hochberg correction of each mutant versus the corresponding wild type. Expression levels were calculated with the ΔΔCt method using ACT8 (AT1G49240) as a standard(Pfaffl 2001) and calibrated by biological replicate 1 (rep1) of Col-0. ...
The study of mutants is one of the best tools for understanding the genetic basis of phenotypes that contribute to adaptation. Oddly, mutant analyses are almost always restricted to single genetic backgrounds and findings therefore can not be easily generalized. A case in point is the key regulator of flowering, FLOWERING LOCUS C ( FLC ), which has been inferred to explain much of the flowering time variation in Arabidopsis thaliana , yet mutants have been examined in very few backgrounds. We have previously established a set of panspecific flc mutants in 62 accessions of A. thaliana (Ruffley et al. 2024). Here, we investigate how genetic background modulates mutant effects on flowering and vegetative traits, as well as on physiology and transcriptomes. Time to onset of flowering in the genome-edited flc lines was reduced by up to 83%, but considerable variation remained. Genetic mapping showed that extremely early flowering in the absence of FLC was mostly explained by natural variation at the known FLC target FT , with additional contribution from loci colocalizing with FLC . Prognostic sequence analyses of accessions did not suggest that extremely-early combinations of engineered flc and natural FT alleles would be deleterious, yet extremely early flowering accessions are not represented in the commonly used collections of A. thaliana accessions. To test whether this discrepancy could be due to sampling bias, we undertook a focused collection effort of wild populations in Southern Italy, which confirmed that extremely early flowering accessions exist in natural populations. Apart from its specific role in flowering time regulation, FLC has pleiotropic effects on other ecophysiological traits such as growth, and these were also dependent on the genetic background, which was further supported by transcriptomic comparisons. Together we conclude that the various roles of FLC have greatly diversified in different genetic backgrounds. Our study provides a proof-of-concept on how analysis of panspecific mutants can reveal the true extent of genetic networks in which a focal gene participates in.
... Table 1 demonstrates the primers' list used for gene amplification. Data was validated using the 2−∆∆Ct formula [16] in the 7500 Fast system Real time PCR (Applied Bio systems, Waltham, Massachusetts, USA). Gene expression and intensity changes were determined by comparative cycle threshold (CT) values, normalized to β-actin. ...
... Table 2 demonstrates the primers' list used for gene amplification. Data were validated using the 2−∆∆ Ct formula [16] in the 7500 Fast system Real time PCR (Applied Bio systems, Waltham, Massachusetts, USA). Gene expression and intensity changes were determined by comparative cycle threshold (CT) values and normalized to β-actin. ...
... RNA pools lacking an RT enzyme from different CL samples, as well as additional negative controls, were included in each qPCR run. The expression level of miRNAs was assessed after their normalization against the Hs_RNU6-2_1 gene, and measurements were undertaken following the Pfaffl method [32]. ...
In cattle, the corpus luteum (CL) is pivotal in maintaining early pregnancy by secreting progesterone. To establish pregnancy, the conceptus produces interferon-τ, preventing luteolysis and initiating the transformation of the CL spurium into a CL verum. Although this transformation is tightly regulated, limited data are available on the expression of microRNAs (miRNAs) during and after this process. To address this gap, we re-analyzed previously published RNA-Seq data of CL from pregnant cows and regressed CL from non-pregnant cows. This analysis identified 44 differentially expressed miRNAs. From this pool, three miRNAs—bta-miR-222-3p, bta-miR-29c, and bta-miR-2411-3p—were randomly selected for relative quantification. Using bovine ovaries (n = 14) obtained from an abattoir, total RNA (including miRNAs) was extracted and converted to cDNA for RT-qPCR. The results revealed that bta-miR-222-3p was downregulated (p = 0.016) in pregnant females compared to non-pregnant cows with regressed CL. However, no differences in miRNA expression were observed between CL of pregnant and non-pregnant cows for bta-miR-29c (p > 0.32) or bta-miR-2411-3p (p > 0.60). In silico prediction approaches indicated that these miRNAs are involved in pathways regulating pregnancy maintenance, such as the VEGF- and FoxO-signaling pathways. Additionally, their biogenesis is regulated by GABPA and E2F4 transcription factors. The validation of selected miRNA expression in the CL during pregnancy by RT-qPCR provides novel insights that could potentially lead to the identification of biomarkers related to CL physiology and pregnancy outcome.
... Genomic DNA was purified as described below. qPCR was used to generate 384 origin (oriC) to terminus (ter) ratios, quantifying the relative amounts of each via the 385 Pfaffl method as described (16,47). qPCR was performed with 20 Table S2 and were used in 392 . ...
DNA replication is regulated by factors that promote or inhibit initiation. In Bacillus subtilis, YabA is a negative regulator of DNA replication initiation while the newly identified kinase CcrZ is a positive regulator. The consequences of under-initiation or over-initiation of DNA replication to genome stability remain unclear. In this work, we measure origin to terminus ratios as a proxy for replication initiation activity. We show that ΔccrZ and several ccrZ alleles under-initiate DNA replication while ablation of yabA or overproduction of CcrZ leads to over-initiation. We find that cells under-initiating DNA replication have few incidents of replication fork stress as determined by low formation of RecA-GFP foci compared with wild type. In contrast, cells over-initiating DNA replication show levels of RecA-GFP foci formation analogous to cells directly challenged with DNA damaging agents. We show that cells under-initiating and over-initiating DNA replication were both sensitive to mitomycin C and that changes in replication initiation frequency cause increased sensitivity to genotoxic stress. With these results, we propose that cells under-initiating DNA replication are sensitive to DNA damage due to a shortage of DNA for repair through homologous recombination. For cells over-initiating DNA replication, we propose that an increase in the number of replication forks leads to replication fork stress which is further exacerbated by chromosomal DNA damage. Together, our study shows that DNA replication initiation frequency must be tightly controlled as changes in initiation influence replication fork fate and the capacity of cells to efficiently repair damage to their genetic material.
... At the end of the amplification phase, a melting curve analysis was performed to confirm the specificity of the PCR product. The relative expression of the gene in each sample versus a control in comparison to GAPDH gene and calculated according to the 2 -ΔΔCt method [31]. ...
HE OBJECTIVE of this work was to elaborate gene expression and serum profile of oxidative/antioxidant and bone markers associated with minerals deficiency and their correlation with mineral deficiency in sheep. The study was carried out on 26 selected diseases cases out of survey on 250 sheep suffered from mineral deficiency compared to 10 apparent healthy control sheep based on clinical examination. Biochemically, there was a significant (P<0.05) decline in Ca, P, Mg, Cu, Zn, Se, Fe, TAC, GPx, SOD, CAT, OC and CP, whereas MDA level was significantly increased (P<0.05). Gene expression of SOD, CAT, GPX1, VDR, CAMK4 and ceruloplasmin genes were significantly down-regulated (P<0.05) in diseased sheep. A contrary tendency was evoked by the gene OXSR1.The variability in studied genes alongside alterations in the serum profile of investigated markers could be a reference guide for limiting the mineral deficiency in sheep through prescribing an efficient nutritional management strategy for sheep flocks that includes the essential macro-and micro aspects, and not depend just on grazing as a primary source of feed to sustain sheep health and output.
... At the end of the amplification phase, a melting curve analysis was performed to confirm the specificity of the PCR product. The relative expression of the gene in each sample versus a control in comparison to GAPDH gene and calculated according to the 2 -ΔΔCt method [31]. ...
THE OBJECTIVE of this work was to elaborate gene expression and serum profile of
oxidative/antioxidant and bone markers associated with minerals deficiency and their
correlation with mineral deficiency in sheep. The study was carried out on 26 selected diseases
cases out of survey on 250 sheep suffered from mineral deficiency compared to 10 apparent healthy
control sheep based on clinical examination. Biochemically, there was a significant (P<0.05) decline
in Ca, P, Mg, Cu, Zn, Se, Fe, TAC, GPx, SOD, CAT, OC and CP, whereas MDA level was
significantly increased (P<0.05). Gene expression of SOD, CAT, GPX1, VDR, CAMK4 and
ceruloplasmin genes were significantly down-regulated (P<0.05) in diseased sheep. A contrary
tendency was evoked by the gene OXSR1.The variability in studied genes alongside alterations in
the serum profile of investigated markers could be a reference guide for limiting the mineral
deficiency in sheep through prescribing an efficient nutritional management strategy for sheep
flocks that includes the essential macro- and micro aspects, and not depend just on grazing as a
primary source of feed to sustain sheep health and output
... After amplification, melting curves were performed on the amplified products, incubating them at 95 • C for 60 s, ramping down to 55 • C, and then increasing temperature to 95 • C at a rate of 0.2 • C/s, measuring fluorescence data continuously. To calculate relative gene expression, efficiency curves were performed for each gene, choosing the most proper calculation method between the equations proposed by Livak and Schimittgen [70] or Pfaffl [71] according to the differences in the amplification efficiency of each one of the analyzed genes. ...
Arsenic compounds have been used as therapeutic alternatives for several diseases including cancer. In the following work, we obtained arsenic nanoparticles (AsNPs) produced by an anaerobic bacterium from the Salar de Ascotán, in northern Chile, and evaluated their effects on the human oral squamous carcinoma cell line OECM-1. Resazurin reduction assays were carried out on these cells using 1–100 µM of AsNPs, finding a concentration-dependent reduction in cell viability that was not observed for the non-tumoral gastric mucosa-derived cell line GES-1. To establish if these effects were associated with apoptosis induction, markers like Bcl2, Bax, and cleaved caspase 3 were analyzed via Western blot, executor caspases 3/7 via luminometry, and DNA fragmentation was analyzed by TUNEL assay, using 100 µM cisplatin as a positive control. OECM-1 cells treated with AsNPs showed an induction of both extrinsic and intrinsic apoptotic pathways, which can be explained by a significant decrease in P-Akt/Akt and P-ERK/ERK relative protein ratios, and an increase in both PTEN and p53 mRNA levels and Bit-1 relative protein levels. These results suggest a prospective mechanism of action for AsNPs that involves a potential interaction with extracellular matrix (ECM) components that reduces cell attachment and subsequently triggers anoikis, an anchorage-dependent type of apoptosis.
... A total of 10 μL of PCR reaction mixture was composed of 10 ng of cDNA, 5 μL of TB Green Premix Ex Taq II (TaKaRa), 5 pmol/L of sense and antisense primers and 1 μL of distilled water. The relative transcription levels of the viral genes were calculated using the Pfaffl equation (Pfaffl 2001) and compared with the TPM values of the venom gland-specific transcriptome data. ...
To identify viruses and compare their abundance levels in the venom glands of hymenopteran species, we conducted venom gland‐specific transcriptome assemblies and analyses of 22 aculeate bees and wasps and identified the RNA genomes of picornaviruses. Additionally, we investigated the expression patterns of viruses in the venom glands over time following capture. Honeybee‐infecting viruses, including the black queen cell virus (BQCV), the deformed wing virus (DWV) and the Israeli acute paralysis virus (IAPV), were highly expressed in the venom glands of Apis mellifera and social wasps. This finding suggests that the venom of bees and wasps is likely to contain these viruses, which can be transmitted horizontally between species through stinger use. Apis mellifera exhibited an increasing pattern of abundance levels for BQCV, DWV, IAPV and Triatovirus , whereas the social wasp Vespa crabro showed increasing abundance levels of IAPV and Triatovirus over different capture periods. This suggests that the venom glands of honeybees and wasps may provide suitable conditions for active viral replication and may be an organ for virus accumulation and transmission. Some viral sequences clearly reflected the phylogeny of aculeate species, implying host‐specific virus evolution. On the other hand, other viruses exhibited unique evolutionary patterns of phylogeny, possibly caused by specific ecological interactions. Our study provides insights into the composition and evolutionary properties of viral genes in the venom glands of certain aculeate bees and wasps, as well as the potential horizontal transmission of these viruses among bee and wasp species.
... OligodT-primed reverse transcription (RT) was performed using 350 ng of total RNA according to the Super Script III kit protocol (Invitrogen). Relative transcript levels were determined by performing RT-PCR (the corresponding gene-specific primer pairs are listed in Table S1) with Power SYBR Green (Applied Biosystems) according to the delta Ct method [20] using a 7500 Real-Time PCR System (Applied Biosystems). GAPDH abundance levels were used to normalize the assay. ...
Background
Neural progenitor cells (NPCs) can be cultivated from developing brains, reproducing many of the processes that occur during neural development. They can be isolated from a variety of animal models, such as transgenic mice carrying mutations in amyloid precursor protein (APP) and presenilin 1 and 2 (PSEN 1 and 2), characteristic of familial Alzheimer’s disease (fAD). Modulating the development of these cells with inflammation-related peptides, such as bradykinin (BK) and its antagonist HOE-140, enables the understanding of the impact of such molecules in a relevant AD model.
Results
We performed a global gene expression analysis on transgenic neurospheres treated with BK and HOE-140. To validate the microarray data, quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed on 8 important genes related to the immune response in AD such as CCL12, CCL5, CCL3, C3, CX3CR1, TLR2 and TNF alpha and Iba-1. Furthermore, comparative analysis of the transcriptional profiles was performed between treatments, including gene ontology and reactome enrichment, construction and analysis of protein-protein interaction networks and, finally, comparison of our data with human dataset from AD patients. The treatments affected the expression levels of genes mainly related to microglia-mediated neuroinflammatory responses, with BK promoting an increase in the expression of genes that enrich processes, biological pathways, and cellular components related to immune dysfunction, neurodegeneration and cell cycle. B2 receptor inhibition by HOE-140 resulted in the reduction of AD-related anomalies caused in this system.
Conclusions
BK is an important immunomodulatory agent and enhances the immunological changes identified in transgenic neurospheres carrying the genetic load of AD. Bradykinin treatments modulate the expression rates of genes related to microglia-mediated neuroinflammation. Inhibiting bradykinin activity in Alzheimer’s disease may slow disease progression.
... Melting curve analyses were performed to 362 confirm single amplicon formation. Relative expression levels were calculated 363 using the Pfaffl method (Pfaffl, 2001). The geometric means of two housekeeping 364 genes ACT2 and UBC21 were used for normalisation (Vandesompele et al., 2002). ...
Floral mimics deceive their pollinators by developing visual and olfactory resemblance to their models. Our knowledge on the diversity of models is expanding rapidly. We report a system in which the flowers exhibit phenotypes similar to aerial litter and deceives an aerial litter specialist beetle to achieve pollination.
We assessed the floral phenology and the effective pollinators of an Australian understorey treelet, Meiogyne heteropetala (Annonaceae). The similarities of morphology, colour and odour between the flowers and co-occurring aerial litter were investigated. The terpene synthase involved in floral scent emission was identified by expression patterns and product profile. The behavioural responses of the pollinator to various odours were assessed using bioassays.
The erotylid beetle Loberus sharpi is the most likely effective pollinator of M. heteropetala , and its eggs were found on the petals of M. heteropetala . Loberus sharpi was exclusively found in aerial litter and M. heteropetala flowers. The morphology and spectral reflectance of the flowers overlap with aerial litter. The floral scent was dominated by monoterpenes, especially 1,8-cineole. The cineole synthase MhCINS was the only highly expressed floral terpene synthase and possessed a highly similar product profile to the floral scent composition. NMDS showed that the volatile composition of M. heteropetala flowers is distinct from other congeners and highly similar to aerial litter, indicating advergence to aerial litter. Visual and odour resemblance, coupled with the deposition of eggs on the flowers, provides evidence that the beetles were deceived into pollinating the flowers. Behavioural experiments showed that the pollinator was attracted to both aerial litter and M. heteropetala flowers. The beetles were also attracted to 1,8-cineole and synthetic mixes of floral odour and MhCINS products. The beetles were unable to distinguish floral scent from MhCINS products, nor from 1,8-cineole, suggesting MhCINS alone sufficed to attract the pollinator olfactorily. The beetles, however, preferred aerial litter over flowers. The beetles likely categorised the flower as a general, but not the most preferred brood substrate.
Synthesis. This study reports the first case of floral mimicry of aerial litter and characterises the biochemical process responsible for olfactory mimicry.
... To calculate the relative expression of genes, we calculated the efficiencies varying between experimental and SmActin's primers. If efficiencies varying by less than 10% the Livak method can be used [38]; if the experimentally established efficiencies vary by more than 10%, a correction should be made using the a Pfaffl mathematical model [39]. Correlation analysis between QRT-PCR results and RNA-Seq expression data was conducted using Python's Pearson correlation method (v 2.7.12). ...
Background
Salvia miltiorrhiza, a well-known traditional Chinese medicine, frequently suffers from replant diseases that adversely affect its quality and yield. To elucidate S. miltiorrhiza’s metabolic adaptations to replant disease, we analyzed its metabolome and transcriptome, comparing normal and replant diseased plants for the first time.
Results
We identified 1,269 metabolites, 257 of which were differentially accumulated metabolites, and identified 217 differentially expressed genes. Integrated transcriptomic and metabolomic analyses revealed a significant up-regulation and co-expression of metabolites and genes associated with plant hormone signal transduction and flavonoid biosynthesis pathways in replant diseases. Within plant hormone signal transduction pathway, plants afflicted with replant disease markedly accumulated indole-3-acetic acid and abscisic acid, correlating with high expression of their biosynthesis-related genes (SmAmidase, SmALDH, SmNCED, and SmAAOX3). Simultaneously, changes in hormone concentrations activated plant hormone signal transduction pathways. Moreover, under replant disease, metabolites in the local flavonoid metabolite biosynthetic pathway were significantly accumulated, consistent with the up-regulated gene (SmHTC1 and SmHTC2). The qRT-PCR analysis largely aligned with the transcriptomic results, confirming the trends in gene expression. Moreover, we identified 10 transcription factors co-expressed with differentially accumulated metabolites.
Conclusions
Overall, we revealed the key genes and metabolites of S. miltiorrhiza under replant disease, establishing a robust foundation for future inquiries into the molecular responses to combat replant stress.
... All reactions were performed in technical duplicates on a Rotor-Gene Q system (Qiagen, Hilden, Germany). Calculations of the relative transcript levels were performed according to the Pfaffl method [51] using the reference genes RSC1 and TAF10 for normalization according to [52]. ...
Background
Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii.
Results
In initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system.
Conclusions
In this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.
... The total number of biological samples was 16 (Control 1 h = 2; Tenmo-AT 10 − 7 M 1 h = 3; Tenmo-AT 10 − 5 M 1 h = 3; Control 24 h = 2; Tenmo-AT 10 − 7 M 24 h = 3; Tenmo-AT 10 − 5 M 24 h = 3). In both cases, the relative expression was calculated based on the primer efficiency using the method described by Pfaffl (2001). ...
... RNA from each biological replicate was extracted using a column-based protocol 53 . 1 µg total RNA was reverse transcribed with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, USA) using an oligo (dT) primer and cDNA was analyzed in a CFX384 Real-Time System Cycler (BioRad, München, Germany). The relative quantification was calculated with the ΔΔCt method using ACT8 (AT1G49240) as a standard 54 and calibrated by biological replicate 1 (rep1 ) of Col-0. ...
Climate change has already caused noticeable changes in species-wide traits, such as the well-documented acceleration of spring flowering. Because the evolutionary past has favored certain combinations of traits, some strategies like fast growth with early flowering that are adaptive today are at odds with other plant resilience strategies such as elevated water use efficiency. We know that the evolution of trait combinations is shaped by genomic constraints, but it is unclear whether and how this is affected by natural selection from climate change. Growing hundreds of Arabidopsis thaliana natural populations under different rainfall regimes revealed opposing natural selection on flowering time and water use efficiency, with strong antagonistic genetic correlations and contrasting causal alleles identified by Genome-Wide Association analyses. Inactivation of the central flowering regulator FLC in multiple, diverse accessions relaxed trait correlations in a genetic background-dependent manner and allowed for the emergence of a novel adaptive trait combination-early flowering and intermediate water use efficiency. Future climates are predicted to escalate conflicts in natural selection among adaptive traits, but our work shows that surprisingly simple genetic changes can help solve these conflicts. Phenotypic trade-offs across individuals within a species, or across species, are common in nature 1. Trade-offs may arise as a result of local natural selection favoring certain trait combinations 2,3 or an 1
... For each primer pair, the amplicon length was verified once by gel electrophoresis and the PCR product was sent to sequencing (Microsynth, Balgach, Switzerland). Ct values were plotted against the natural logarithm of the template concentration and PCR efficiencies were calculated from the slope of the regression line according to the equation E = 10 [−1/slope] [17][18][19]. ...
Background
Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design.
Results
ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets.
Conclusion
ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.
... To ensure the reliability of the results, each experimental sample included three biological replicates, and each biological replicate was repeated three times. The expression level of the target genes was quantitatively analyzed using the 2 ÀDDCT method (Pfaffl, 2001). ...
Acacetin, a flavonoid compound, possesses a wide range of pharmacological effects, including antimicro-bial, immune regulation, and anticancer effects. Some key steps in its biosynthetic pathway were largely unknown in flowering plants. Here, we present the first haplotype-resolved genome of Chrysanthemum indicum, whose dried flowers contain abundant flavonoids and have been utilized as traditional Chinese medicine. Various phylogenetic analyses revealed almost equal proportion of three tree topologies among three Chrysanthemum species (C. indicum, C. nankingense, and C. lavandulifolium), indicating that frequent gene flow among Chrysanthemum species or incomplete lineage sorting due to rapid speciation might contribute to conflict topologies. The expanded gene families in C. indicum were associated with oxidative functions. Through comprehensive candidate gene screening, we identified five flavonoid O-methyltransferase (FOMT) candidates, which were highly expressed in flowers and whose expressional levels were significantly correlated with the content of acacetin. Further experiments validated two FOMTs (CI02A009970 and CI03A006662) were capable of catalyzing the conversion of apigenin into acacetin, and these two genes are possibly responsible acacetin accumulation in disc florets and young leaves, respectively. Furthermore, combined analyses of ancestral chromosome reconstruction and phylogenetic trees revealed the distinct evolutionary fates of the two validated FOMT genes. Our study provides new insights into the biosynthetic pathway of flavonoid compounds in the Asteraceae family and offers a model for tracing the origin and evolutionary routes of single genes. These findings will facilitate in vitro biosynthetic production of flavonoid compounds through cellular and metabolic engineering and expedite molecular breeding of C. indicum cultivars.
... The quantification was performed using the comparative Ct (2 −∆∆Ct ) method [85]. Several time points were considered for in vitro and in vivo assays, including one before infection kinetics began (time 0 or control). ...
The innate immune response in Salmo salar, mediated by pattern recognition receptors (PRRs), is crucial for defending against pathogens. This study examined DDX41 protein functions as a cytosolic/nuclear sensor for cyclic dinucleotides, RNA, and DNA from invasive intracellular bacteria. The investigation determined the existence, conservation, and functional expression of the ddx41 gene in S. salar. In silico predictions and experimental validations identified a single ddx41 gene on chromosome 5 in S. salar, showing 83.92% homology with its human counterpart. Transcriptomic analysis in salmon head kidney confirmed gene transcriptional integrity. Proteomic identification through mass spectrometry characterized three unique peptides with 99.99% statistical confidence. Phylogenetic analysis demonstrated significant evolutionary conservation across species. Functional gene expression analysis in SHK-1 cells infected by Piscirickettsia salmonis and Renibacterium salmoninarum indicated significant upregulation of DDX41, correlated with increased proinflammatory cytokine levels and activation of irf3 and interferon signaling pathways. In vivo studies corroborated DDX41 activation in immune responses, particularly when S. salar was challenged with P. salmonis, underscoring its potential in enhancing disease resistance. This is the first study to identify the DDX41 pathway as a key component in S. salar innate immune response to invading pathogens, establishing a basis for future research in salmonid disease resistance.
... Real-time polymerase chain reaction (PCR) for specific miRNAs was performed using Ago2/Loqs (internal control), for which the primers were designed as per Saito et al. (42) and Taqman real-time PCR probes for miR-34 supplied by Applied Biosystems, as per the manufacturer's protocol on Roche Light Cycler480. Data were extracted and analysed manually using the method described by Pfaffl (43). ...
... The list of primers used for genes of interest are given in the table. The comparative C T method (ΔΔCT method) was used to determine relative expression of target gene to the house keeping gene [58]. ...
Mitochondria react to infection with sub-lethal signals in the apoptosis pathway. Mitochondrial signals can be inflammatory but mechanisms are only partially understood. We show that activation of the caspase-activated DNase (CAD) mediates mitochondrial pro-inflammatory functions and substantially contributes to host defense against viral infection. In cells lacking CAD, the pro-inflammatory activity of sub-lethal signals was reduced. Experimental activation of CAD caused transient DNA-damage and a pronounced DNA damage response, involving major kinase signaling pathways, NF-κB and cGAS/STING, driving the production of interferon, cytokines/chemokines and attracting neutrophils. The transcriptional response to CAD-activation was reminiscent of the reaction to microbial infection. CAD-deficient cells had a diminished response to viral infection. Influenza virus infected CAD-deficient mice displayed reduced inflammation in lung tissue, higher viral titers and increased weight loss. Thus, CAD links the mitochondrial apoptosis system and cell death caspases to host defense. CAD-driven DNA damage is a physiological element of the inflammatory response to infection.
... 18S rRNA was used as a reference control. The fold changes in gene expression were determined using 2ΔΔCt 51 . The reported data was the mean of three replicates. ...
Acclimation to crop niches for thousands of years has made indigenous rice cultivars better suited for stress-prone environments. Still, their response to UV-B resiliency is unknown. 38 rice landraces were grown in cemented pots in a randomised block design with three replicates under open field conditions in Sambalpur University in the wet season of 2022. Half of the plants in each of the cultivars were administered UV-B radiation at the panicle emergence stage in an adjustable UV-B chamber permitting sunlight, and the effects of the stress on various morpho-physiological features, such as spikelet sterility, flag leaf photosynthetic and flavonoid pigment contents, and lipid peroxidation activities, were estimated for calibration of stress resistance. The experiment identified Swarnaprabha and Lalkain as the most sensitive and resilient to stress respectively, and the differential response between them was further revealed in the expression of genes related to UV-B sensitivity. Subject to the stress, Swarnaprabha exhibited symptoms of injuries, like leaf burns, and a higher loss of various photosynthetic parameters, such as pigment contents, SPAD and Fv/Fm, ETR and qP values, while NPQ increased only in Lalkain. Exposure to UV-B increased the total phenolic and flavonoid contents in Lalkain while depressing them in Swarnaprabha. Such an effect amounted to a higher release of fluorescent energy in the latter. The levels of expression of gene families controlling flavonoid activation and UV-B signal transduction, such as OsWRKY, OsUGT, OsRLCK, OsBZIP, OsGLP, and CPD photolyase were similar in both the cultivars in the control condition. However, exposure to UV-B stress overexpressed them in resilient cultivars only. The magnitude of expression of the genes and the impact of the stress on photosynthetic parameters, phenolic compounds and pubescent hair structure at the panicle emergence stage could be valid indicators among indigenous rice for UV-B tolerance.
... For each RT-qPCR experiment, using beta-actin as a transcript normalizer, the relative expression of a target gene was calculated using the ΔΔCT technique. 15 The raw data were produced using Rotor-Gene Q series software version 2.3.1. ...
BACKGROUND
Flystrike, primarily caused by Lucilia cuprina, is a major health and welfare issue for sheep wool industries. Current chemical‐based controls can have limited effectiveness due to the emergence of resistance in the parasite. RNA interference (RNAi), which uses double‐stranded RNA (dsRNA) as a trigger molecule, has been successfully investigated for the development of innovative pest control strategies. Although RNAi offers great potential, the efficient identification, selection of target genes and delivery of dsRNA represent challenges to be overcome for the successful application of RNAi for control of L. cuprina.
RESULTS
A primary L. cuprina (blowfly) embryo cell line (BFEC) was established and confirmed as being derived from L. cuprina eggs by PCR and amplicon sequencing. The BFECs were successfully transfected with plasmids and messenger RNA (mRNA) expressing fluorescent reporter proteins and dsRNA using lipid‐based transfection reagents. The transfection of dsRNA into BEFC in this study suggested decreased mRNA levels of target gene expression, which suggested RNAi‐mediated knockdown. Three of the dsRNAs identified in this study resulted in reductions of in target gene mRNA levels in BFEC and loss of biological fitness by L. cuprina larvae in a feeding bioassay.
CONCLUSION
This study confirms that the novel BFEC cell line can be used to improve the efficacy of dsRNA‐mediated screening to accelerate the identification of potential target genes in the development of RNAi mediated control approaches for L. cuprina. The research models established in this study are encouraging with respect to the use of RNAi as a blowfly control method, however further improvement and validation are required for field applicationsnot prefect, and could be ongoing developing. © 2024 Society of Chemical Industry.
... The thermal cycling conditions used in qPCR for 40 cycles are presented in the Table 3. The cycle threshold (C t ) values obtained for the target gene and reference gene (beta-actin) were used to obtain relative quantification (R) using the formula ΔΔct given by Pfaffl (2001). The treatment diet T 5 formulated as per ICAR recommendations served as control group. ...
Nutrient supply regulates overall body growth directly or indirectly through its influence on regulatory factors optimizing nutrient requirements becomes crucial before embarking on genetic improvements. Hence this study addresses this gap by evaluating the effect of feeding varying energy and crude protein levels on growth performance and gene expression related to the growth of indigenous Siruvidai chicken from 0 to 12 weeks. A 360-day-old straight-run Siruvidai chick were randomly distributed into six experimental groups with three replicates of each 20 chicks. The birds were fed corn-soy-based diets formulated with two levels of energy (2500 and 2700 kcal ME/kg) each with three levels of crude protein (16, 18, and 20%) during the brooder stage (0–12 weeks) in 2 × 3 factorial design. Results revealed that there was no significant effect on the energy and protein interaction levels on average feed intake, body weight gain and feed conversion ratio in Siruvidai chicken at 12 weeks. The results showed significantly (P < 0.05) lower feed intake in 18% protein fed groups and significantly (P < 0.01) lower feed intake in higher energy 2700 kcal ME/kg fed groups. A better feed conversion ratio (4.06 and 4.21) was observed on the effect of protein levels in bird diets with 18% and 20% protein fed groups. The Growth Hormone (GH) and Myostatin (MSTN) gene expression were significantly (P < 0.01) higher in 16% CP and 2500 kcal ME/kg in hepatic tissue. The high protein and low energy diet up-regulated the Insulin-like Growth Factor-1 (IGF-1) gene expression in hepatic tissue. The study concluded that Siruvidai chicken fed with 18% crude protein and 2500 kcal ME/kg is optimum for 0–12 weeks of age.
... A25776) on the 7500 Fast Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific, Nepean, ON, Canada). Gene expression was normalized to GAPDH, and fold change was calculated relative to the mock-treated samples for each gene using the Pfaffl method [22]. ...
Oncolytic virotherapy, using viruses such as vesicular stomatitis virus (VSVΔ51) and Herpes Simplex Virus-1 (HSV-1) to selectively attack cancer cells, faces challenges such as cellular resistance mediated by the interferon (IFN) response. Dimethyl fumarate (DMF) is used in the treatment of multiple sclerosis and psoriasis and is recognized for its anti-cancer properties and has been shown to enhance both VSVΔ51 and HSV-1 oncolytic activity. Tepilamide fumarate (TPF) is a DMF analog currently undergoing clinical trials for the treatment of moderate-to-severe plaque psoriasis. The aim of this study was to evaluate the potential of TPF in enhancing the effectiveness of oncolytic viruses. In vitro, TPF treatment rendered 786-0 carcinoma cells more susceptible to VSVΔ51 infection, leading to increased viral replication. It outperformed DMF in both increasing viral infection and increasing the killing of these resistant cancer cells and other cancer cell lines tested. Ex vivo studies demonstrated TPF’s selective boosting of oncolytic virus infection in cancer cells without affecting healthy tissues. Effectiveness was notably high in pancreatic and ovarian tumor samples. Our study further indicates that TPF can downregulate the IFN pathway through a similar mechanism to DMF, making resistant cancer cells more vulnerable to viral infection. Furthermore, TPF’s impact on gene therapy was assessed, revealing its ability to enhance the transduction efficiency of vectors such as lentivirus, adenovirus type 5, and adeno-associated virus type 2 across various cell lines. This data underscore TPF’s potential role in not only oncolytic virotherapy but also in the broader application of gene therapy. Collectively, these findings position TPF as a promising agent in oncolytic virotherapy, warranting further exploration of its therapeutic potential.
... Quantitative reverse transcriptase PCR was conducted with the CFX384TM real-time system (Bio-Rad, Germany) and Go Taq qPCR Master Mix SybrGreen I (Promega) using the primers listed in Supplemental Table 2. UBQ2 was used as a reference gene to normalize relative expression levels of all tested genes. Relative expression was calculated according to Pfaffl (2001). ...
... To obtain the Arabidopsis thaliana homologs, Blastp and the TAIR10 library from Ensembl were used, with the options num_alignments 1 and e-value 1e − 08 . AgriGO V2.0 [92] was used for gene ontology enrichment for the best Arabidopsis homologs (FDR ≤ 0.05). The GO terms were manually collapsed based on the analogous description and the set of genes they contained. ...
Background
The periderm is basic for land plants due to its protective role during radial growth, which is achieved by the polymers deposited in the cell walls. In most trees, like holm oak, the first periderm is frequently replaced by subsequent internal periderms yielding a heterogeneous outer bark made of a mixture of periderms and phloem tissues, known as rhytidome. Exceptionally, cork oak forms a persistent or long-lived periderm which results in a homogeneous outer bark of thick phellem cell layers known as cork. Cork oak and holm oak distribution ranges overlap to a great extent, and they often share stands, where they can hybridize and produce offspring showing a rhytidome-type bark.
Results
Here we use the outer bark of cork oak, holm oak, and their natural hybrids to analyse the chemical composition, the anatomy and the transcriptome, and further understand the mechanisms underlying periderm development. We also include a unique natural hybrid individual corresponding to a backcross with cork oak that, interestingly, shows a cork-type bark. The inclusion of hybrid samples showing rhytidome-type and cork-type barks is valuable to approach cork and rhytidome development, allowing an accurate identification of candidate genes and processes. The present study underscores that abiotic stress and cell death are enhanced in rhytidome-type barks whereas lipid metabolism and cell cycle are enriched in cork-type barks. Development-related DEGs showing the highest expression, highlight cell division, cell expansion, and cell differentiation as key processes leading to cork or rhytidome-type barks.
Conclusion
Transcriptome results, in agreement with anatomical and chemical analyses, show that rhytidome and cork-type barks are active in periderm development, and suberin and lignin deposition. Development and cell wall-related DEGs suggest that cell division and expansion are upregulated in cork-type barks whereas cell differentiation is enhanced in rhytidome-type barks.
... Post-amplification melting-curve analyses were performed to confirm reaction specificity. The expression levels of the target genes of the different treatments were normalized to the actin housekeeping gene and compared using a relative quantification method [39]. Real-time data are presented as the fold change in expression levels. ...
Background
Type I interferons (IFN-I)—a group of cytokines with immunomodulatory, antiproliferative, and antiviral properties—are widely used as therapeutics for various cancers and viral diseases. Since IFNs are proteins, they are highly susceptible to degradation by proteases and by hydrolysis in the strong acid environment of the stomach, and they are therefore administered parenterally. In this study, we examined whether the intestinal bacterium, enteropathogenic Escherichia coli (EPEC), can be exploited for oral delivery of IFN-Is. EPEC survives the harsh conditions of the stomach and, upon reaching the small intestine, expresses a type III secretion system (T3SS) that is used to translocate effector proteins across the bacterial envelope into the eukaryotic host cells.
Results
In this study, we developed an attenuated EPEC strain that cannot colonize the host but can secrete functional human IFNα2 variant through the T3SS. We found that this bacteria-secreted IFN exhibited antiproliferative and antiviral activities similar to commercially available IFN.
Conclusion
These findings present a potential novel approach for the oral delivery of IFN via secreting bacteria.
... qRT-PCR was carried out on a fluorescence quantitative PCR system (Analytikjena qTOWER3, Jena, Germany) with the following conditions: 95°C for 30 s; 40 cycles of 95°C for 10 s and 54°C for 30 s; and 72°C for 15 s. The relative gene expression levels were calculated using the DDCT method (Pfaffl 2001), and each expression level represented the mean of three biological replicates. ...
Brachypodium distachyon has emerged as a new model for the structural and functional genomics of temperate grasses. Somatic embryo culture serves as an effective method for the genetic transformation of B. distachyon. However, the role of regulatory genes in inducing somatic embryogenesis through endogenous hormone modulation in B. distachyon remains unexplored. This study investigated the endogenous hormone levels and transcriptome data of embryogenic calli (EC) derived from both mature and immature B. distachyon embryos. Our findings indicate that higher levels of IAA and GAs promote the formation of mature embryo-produced compact embryogenic calli (MEC), whereas higher tZR and lower ABA levels promote the development of immature embryo-produced compact embryogenic calli (IEC). The differential expression of genes involved in the biosynthesis and signal transduction of auxin (YUCCA,ALDH, AUX/IAA), trans-zeatin riboside (CKX), gibberellin (KAO, GA3ox), and abscisic acid (AAO3, SnRK2) influences the endogenous hormone concentrations in MEC and IEC of B. distachyon. Specifically, increased expression of AUX/IAA is linked to MEC formation. Thus, the differential expression of genes associated with the AUX, CK, GA, and ABA biosynthesis and signal transduction pathways may modulate endogenous hormone levels, steering the differentiation of MEC and IEC in B. distachyon. Keywords Brachypodium distachyon·embryogenic calli· differentially expressed genes·endogenous hormone
... The RNA was eluted with 50 μL of nuclease-free water, and RNA was quantified with a Nanodrop OneC (Thermo Fisher Scientific, Madison, WI, USA). Then, expression of ZO-1, occludin, claudin-1, claudin-2, and HPRT1 (internal standard) genes was assessed by RT-PCR 40 . Complementary DNA synthesis and RT-PCR were performed with a Thermal Cycler Dice Real Time System TP970 (Takara) and One ...
Heat-killed Lactiplantibacillus plantarum L-137 (HK L-137) has been suggested to enhance the intestinal barrier in obese mice, leading to improvement of metabolic abnormalities and adipose tissue inflammation, and in healthy humans with overweight, leading to improvement of systemic inflammation. However, its detailed mechanism of action has not been clarified. Therefore, this study investigated the effects of HK L-137 on the permeability of rat small intestinal epithelial IEC-6 cells, tight junction-related gene and protein expression and localization, and intracellular signaling pathways involved in barrier function. Treatment of IEC-6 cells with HK L-137 for 26 h significantly reduced the permeability to fluorescein isothiocyanate-dextran (FD-4). HK L-137 also increased gene and protein expression of zonula occludens-1 (ZO-1), an important tight junction protein, without affecting the localization. Furthermore, inhibition of the extracellular signal-regulated kinase (ERK)1/2 pathway in IEC-6 cells canceled the HK L-137-related reduction in permeability to FD-4. Phosphorylation of ERK in IEC-6 cells was induced 15 min after the addition of HK L-137. These results suggest that HK L-137 reduces intestinal permeability partly through activating the ERK pathway and increasing expression of the ZO-1 gene and protein. Enhancement of intestinal barrier function with HK L-137 might be effective in preventing and treating leaky gut, for which no specific therapeutic tool has been established.
... where E(target) and E(control) are the qPCR efficiencies of the target (i.e., telomere, rDNA, and mtDNA) and the single copy gene respectively, and C t GS and C t SAMPLE are the critical cycle thresholds for the golden standard and sample DNAs respectively 117,118 . ...
Many fisheries exert directional selection on traits such as body size and growth rate. Whether directional selection impacts regions of the genome associated with traits related to growth is unknown. To address this issue, we characterised copy number variation in three regions of the genome associated with cell division, (1) telomeric DNA, (2) loci transcribed as ribosomal RNA (rDNA), and (3) mitochondrial DNA (mtDNA), in three selection lines of zebrafish reared at three temperatures (22 °C, 28 °C, and 34 °C). Selection lines differed in (1) the direction of selection (two lines experienced directional selection for large or small body size) and (2) whether they experienced any directional selection itself. Lines that had experienced directional selection were smaller, had lower growth rate, shorter telomeres, and lower rDNA copy number than the line that experiencing no directional selection. Neither telomere length nor rDNA copy number were affected by temperature. In contrast, mtDNA content increased at elevated temperature but did not differ among selection lines. Though directional selection impacts rDNA and telomere length, direction of such selection did not matter, whereas mtDNA acts as a stress marker for temperature. Future work should examine the consequences of these genomic changes in natural fish stocks.
... At least three independent experiments with triplicates for in vitro studies (final samples ≥9) and more than five mice/group/sex were considered. The abundance of mRNA was calculated based on the Pfaffl method and genespecific efficiencies 56 , relative to the geometric means of the 2 housekeeping genes. Four different housekeeping genes (Beta actin, GAPDH, HMBS, and HMBeta2) were tested in HepG2 cell studies. ...
Nesfatin-1 (NESF-1) has been shown to modulate lipid metabolism. We have identified a nesfatin-1-like-peptide (NLP) processed from a related precursor nucleobindin 1 (NUCB1). Here we determined if NLP, like NESF-1, regulates lipid accumulation in vitro, and tested if the disruption of nucb1 gene affects hepatic lipid metabolism genes in mice. Hepatocytes (HepG2/C3A cells) express NLP and NESF-1 and both peptides significantly reduced lipogenic enzyme mRNAs and enhanced beta-oxidation enzyme mRNAs. Lipid contents in oleic acid induced HepG2/C3A cells were attenuated by NESF-1 and NLP. The inhibitory effect on cellular lipid content was blocked by compound C, an inhibitor of AMPK. The disruption of nucb1 gene affected lipid metabolism-related enzyme mRNAs, endogenous nucb2 mRNA and AMPK phosphorylation. The lipid-lowering effects identified here highlights the potential of nucleobindins and peptides processed from them to address lipid disorders, and its possible benefits in metabolic disease management.
... The relative quantification value of each target gene was expressed using the Ct comparative method with the 2 -∆∆Ct formula. The β2M reference gene was chosen after GeNorm TM analysis (29,30,31) . ...
Ganoderma lucidum (a mushroom used in traditional Chinese medicine) compounds may attenuate aging-related physiological changes and restore normal immunity. However, studies on the physiological effects of Ganoderma lucidum dry extract food supplements are few. Therefore, here, we aimed to investigate the effects of Ganoderma lucidum dry extract food supplement on the lymphocyte function of older women. This was a double-blind clinical trial (n = 60) with a final 39 older volunteers, divided into two groups, Ganoderma lucidum ( n = 23) and placebo (n = 16). The Ganoderma lucidum group received 2,000 mg/day of Ganoderma lucidum dry extract for 8 weeks. We used flow cytometry to determine the lymphocyte profile. CD4 ⁺ lymphocyte gene expression was evaluated by real-time PCR. We observed that in the Ganoderma lucidum group, concanavalin A (ConA) stimulation increased lymphocyte proliferation. Further, we observed an increase in expression of FOXP3, TGF-β, IL-10, IL-6, RORγ, GATA-3, and IFN-γ genes in the Ganoderma lucidum group. Furthermore, in the Ganoderma lucidum group, ionomycin and PMA stimulation led to decrease in Th17 ⁺ cells and increase in Th2 ⁺ cells. Thus, in older women, Ganoderma lucidum regulates T lymphocyte function leading to a predominant anti-inflammatory action but does not induce T lymphocyte proliferation through CD28 signaling pathway.
... SYBR Green reagent (TaKaRa, Japan) was used in an ABI Prism 7700 Real-Time PCR equipment (Applied Biosystems, USA) to amplify the mRNA by Real-Time Quantitative PCR (RT-qPCR). The relative gene expression was normalized to the internal control, GADPH, using the 2-ΔΔCt formula (30). The primers for Mouse SLIT3, Mouse RhoA, Rat SLIT3, Rat Collagen I, Rat Collagen III, Rat ACTA2 (α-SMA), Rat Fibronectin, Rat CTGF, and Rat GAPDH were designed by the NCBI Primer-Blast Tool (https://www.ncbi.nlm.nih. ...
Objective(s)
Slit guidance ligand 3 (SLIT3) has been identified as a potential therapeutic regulator against fibroblast activity and fibrillary collagen production in an autocrine manner. However, this research aims to investigate the potential role of SLIT3 in cardiac fibrosis and fibroblast differentiation and its underlying mechanism.
Materials and Methods
C57BL/6 mice (male, 8-10 weeks, n=47) were subcutaneously infused with Ang II (2.0 mg/kg/day) for 4 weeks. One to two-day-old Sprague-Dawley (SD) rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (60 mg/kg) and ketamine (50 mg/kg) and the cardiac fibroblast was isolated aseptically. The mRNA and protein expression were analyzed using RT-qPCR and Western blotting.
Results
The SLIT3 expression level was increased in Ang II-induced mice models and cardiac fibroblasts. SLIT3 significantly increased migrated cells and α-smooth muscle actin (α-SMA) expression in cardiac fibroblasts. Ang II-induced increases in mRNA expression of collagen I (COL1A1), and collagen III (COL3A1) was attenuated by SLIT3 inhibition. SLIT3 knockdown attenuated the Ang II-induced increase in mRNA expression of ACTA2 (α-SMA), Fibronectin, and CTGF. SLIT3 suppression potentially reduced DHE expression and decreased malondialdehyde (MDA) content, and the superoxide dismutase (SOD) and catalase (CAT) levels were significantly increased in cardiac fibroblasts. Additionally, SLIT3 inhibition markedly decreased RhoA and ROCK1 protein expression, whereas ROCK inhibitor Y-27632 (10 μM) markedly attenuated the migration of cardiac fibroblasts stimulated by Ang II and SLIT3.
Conclusion
The results speculate that SLIT3 could significantly regulate cardiac fibrosis and fibroblast differentiation via the RhoA/ROCK1 signaling pathway.
... Primer details and amplification conditions are listed in Table S2. Data were analyzed as ΔΔCT correcting for the actual efficiencies of the primers used [57]. ...
Stress affects the brain and alters its neuroarchitecture and function; these changes can be severe and lead to psychiatric disorders. Recent evidence suggests that astrocytes and microglia play an essential role in the stress response by contributing to the maintenance of cerebral homeostasis. These cells respond rapidly to all stimuli that reach the brain, including stressors. Here, we used a recently validated rodent model of post-traumatic stress disorder in which rats can be categorized as resilient or vulnerable after acute inescapable footshock stress. We then investigated the functional, molecular, and morphological determinants of stress resilience and vulnerability in the prefrontal cortex, focusing on glial and neuronal cells. In addition, we examined the effects of a single subanesthetic dose of ketamine, a fast-acting antidepressant recently approved for the treatment of resistant depression and proposed for other stress-related psychiatric disorders. The present results suggest a prompt glial cell response and activation of the NF-κB pathway after acute stress, leading to an increase in specific cytokines such as IL-18 and TNF-α. This response persists in vulnerable individuals and is accompanied by a significant change in the levels of critical glial proteins such as S100B, CD11b, and CX43, brain trophic factors such as BDNF and FGF2, and proteins related to dendritic arborization and synaptic architecture such as MAP2 and PSD95. Administration of ketamine 24 h after the acute stress event rescued many of the changes observed in vulnerable rats, possibly contributing to support brain homeostasis. Overall, our results suggest that pivotal events, including reactive astrogliosis, changes in brain trophic factors, and neuronal damage are critical determinants of vulnerability to acute traumatic stress and confirm the therapeutic effect of acute ketamine against the development of stress-related psychiatric disorders.
... Constitutively expressed CYCLO and ACTIN genes were used as reference genes to normalize qRT-PCR results. The relative expression levels were calculated from the threshold cycles (Ct) values and the primer efficiencies by the Pfaffl method [90]. Each PCR analysis was conducted on three biological replicates (each biological replicate was a mixture of the roots of two plants), and each PCR reaction was repeated twice. ...
The European “Green Deal” policies are shifting toward more sustainable and environmentally conscious agricultural practices, reducing the use of chemical fertilizer and pesticides. This implies exploring alternative strategies. One promising alternative to improve plant nutrition and reinforce plant defenses is the use of beneficial microorganisms in the rhizosphere, such as “Plant-growth-promoting rhizobacteria and fungi”. Despite the great abundance of iron (Fe) in the Earth’s crust, its poor solubility in calcareous soil makes Fe deficiency a major agricultural issue worldwide. Among plant promoting microorganisms, the yeast Debaryomyces hansenii has been very recently incorporated, for its ability to induce morphological and physiological key responses to Fe deficiency in plants, under hydroponic culture conditions. The present work takes it a step further and explores the potential of D. hansenii to improve plant nutrition and stimulate growth in cucumber plants grown in calcareous soil, where ferric chlorosis is common. Additionally, the study examines D. hansenii’s ability to induce systemic resistance (ISR) through a comparative relative expression study by qRT-PCR of ethylene (ET) biosynthesis (ACO1), or ET signaling (EIN2 and EIN3), and salicylic acid (SA) biosynthesis (PAL)-related genes. The results mark a significant milestone since D. hansenii not only enhances nutrient uptake and stimulates plant growth and flower development but could also amplify induced systemic resistance (ISR). Although there is still much work ahead, these findings make D. hansenii a promising candidate to be used for sustainable and environmentally friendly integrated crop management.
... For RNA isolation from PMCC, cell lysates were directly dissolved in RLT buffer and continued as described earlier [33]. The specific forward and reverse primers and Taqman probes (TIB Molbiol, Berlin, Germany) used in this study are described in Table 2. Gene expressions were normalized to the internal control TATA box binding protein using a modified version of the ∆∆CT method [54]. The means of control animal samples were set to 1. Table 2. Primers and probes used for qRTPCR. ...
Stress exposure worsens allergic inflammatory diseases substantially. Mast cells (MCs) play a key role in peripheral immune responses to neuroendocrine stress mediators such as nerve growth factor (NGF) and substance P (SP). Mast cell proteases (MCPs) and cholinergic factors (Chrna7, SLURP1) were recently described to modulate MC stress response. We studied MCPs and Chrna7/SLURP1 and their interplay in a mouse model for noise induced stress (NiS) and atopic dermatitis-like allergic inflammation (AlD) and in cultured MC lacking Chrna7. We found that the cholinergic stress axis interacts with neuroendocrine stress mediators and stress-mediator cleaving enzymes in AlD. SP-cleaving mMCP4+ MC were upregulated in AlD and further upregulated by stress in NiS+AlD. Anti-NGF neutralizing antibody treatment blocked the stress-induced upregulation in vivo, and mMCP4+ MCs correlated with measures of AlD disease activity. Finally, high mMCP4 production in response to SP depended on Chrna7/SLURP1 in cultured MCs. In conclusion, mMCP4 and its upstream regulation by Chrna7/SLURP1 are interesting novel targets for the treatment of allergic inflammation and its aggravation by stress.
... The relative messenger RNA (mRNA) levels of the target genes were calculated using the 2 − Ct method (Schmittgen & Livak, 2008). The homologous RT-qPCR efficiencies (E) were calculated according to the equation: E = (10 [−1/slope] − 1) × 100 (Pfaffl, 2001). ...
Vitellogenin receptor (VgR) plays a crucial role in oogenesis by mediating endocytosis of vitellogenin and a portion of the yolk proteins in many insect species. However, the function of VgR in minute parasitoid wasps is largely unknown. Here, we applied Trichogramma dendrolimi , a minute egg parasitoid, as a study model to investigate the function of VgR in parasitoids. We developed RNA interference (RNAi) methods based on microinjection of prepupae in T. dendrolimi . RNAi employs nanomaterial branched amphipathic peptide capsules (BAPC) as a carrier for double‐stranded RNA (dsRNA), significantly enhancing delivery efficiency. Also, artificial hosts without medium were used to culture the injected prepupae in vitro . Utilizing these methods, we found that ovarian growth was disrupted after knockdown of TdVgR , as manifested by the suppressed development of the ovariole and the inhibition of nurse cell internalization by oocytes. Also, the initial mature egg load in the ovary was significantly reduced. Notably, the parasitic capacity of the female adult with ovarian dysplasia was significantly decreased, possibly resulting from the low availability of mature eggs. Moreover, ovarian dysplasia in T. dendrolimi caused by VgR deficiency are conserved despite feeding on different hosts. The results confirmed a critical role of TdVgR in the reproductive ability of T. dendrolimi and provided a reference for gene functional studies in minute insects.
... Cycle threshold values for the gene of interest were normalized in respect to the three reference genes and the geometric mean of expression was calculated. The relative expression was determined using the ΔΔCt mathematical model corrected for the PCR efficiency (E) [38]. The relative quantification was estimated in comparison with either "crown-1DAG RNAseq " or "roots-1DAG" sample. ...
Background
Roots play an important role during plant growth and development, ensuring water and nutrient uptake. Understanding the mechanisms regulating their initiation and development opens doors towards root system architecture engineering.
Results
Here, we investigated by RNA-seq analysis the changes in gene expression in the barley stem base of 1 day-after-germination (DAG) and 10DAG seedlings when crown roots are formed. We identified 2,333 genes whose expression was lower in the stem base of 10DAG seedlings compared to 1DAG seedlings. Those genes were mostly related to basal cellular activity such as cell cycle organization, protein biosynthesis, chromatin organization, cytoskeleton organization or nucleotide metabolism. In opposite, 2,932 genes showed up-regulation in the stem base of 10DAG seedlings compared to 1DAG seedlings, and their function was related to phytohormone action, solute transport, redox homeostasis, protein modification, secondary metabolism. Our results highlighted genes that are likely involved in the different steps of crown root formation from initiation to primordia differentiation and emergence, and revealed the activation of different hormonal pathways during this process.
Conclusions
This whole transcriptomic study is the first study aiming at understanding the molecular mechanisms controlling crown root development in barley. The results shed light on crown root emergence that is likely associated with a strong cell wall modification, death of the cells covering the crown root primordium, and the production of defense molecules that might prevent pathogen infection at the site of root emergence.
... Rpl32 is a common housekeeping gene[150] and was chosen here as a normalization gene based on the minimal non-significant change (log2 fold change = 0.08) in rpl32 expression observed in the Illumina RNA-seq between Nab2 pex41 and Nab2 ex3 larval brains. Threshold (CT) values for each sample were then averaged and the 2 -DCT method[158,160] was used for relative quantification. Standard error values for the relative expression of each gene were calculated from corresponding 2 -DCT values from each biological replicate. ...
The regulation of cell-specific gene expression patterns during development requires the coordinated actions of hundreds of proteins, including transcription factors, processing enzymes, and many RNA binding proteins (RBPs). RBPs often become associated with a nascent transcript immediately after its production and are uniquely positioned to coordinate concurrent processing and quality control steps. Since RNA binding proteins can regulate multiple post-transcriptional processing steps for many mRNA transcripts, mutations within RBP-encoding genes often lead to pleiotropic effects that alter the physiology of multiple cell types. Thus, identifying the mRNA processing steps where an RBP functions and the effects of RBP loss on gene expression patterns can provide a better understanding of both tissue physiology and mechanisms of disease.
In the current study, we have investigated the coordination of mRNA splicing and polyadenylation facilitated by the Drosophila RNA binding protein Nab2, an evolutionary conserved ortholog of human ZC3H14. ZC3H14 loss in human patients has previously been linked to alterations in nervous system function and disease. Both fly Nab2 and vertebrate ZC3H14 bind to polyadenosine RNA and have been implicated in the control of poly(A) tail length. Interestingly, we show that fly Nab2 functionally interacts with components of the spliceosome, suggesting that this family of RNA biding proteins may also regulate alternative splicing of mRNA transcripts. Using RNA-sequencing approaches, we show that Nab2 loss causes widespread changes in alternative splicing and intron retention. These changes in splicing cause alterations in the abundance of protein isoforms encoded by the affected transcripts and may contribute to phenotypes, such as decreases in viability and alterations in brain morphology, observed in Nab2 null flies. Overall, these studies highlight the importance of RNA binding proteins in the coordination of post-transcriptional gene expression regulation and potentially identify a class of proteins that can coordinate multiple processing events for specific mRNA transcripts.
Author Summary
Although most cells in a multicellular organism contain the same genetic material, each cell type produces a set of RNA molecules and proteins that allows it to perform specific functions. Protein production requires that a copy of the genetic information encoded in a cell’s DNA first be copied into RNA. Then the RNA is often processed to remove extra sequences and the finalized RNA can be used to create a particular type of protein. Our work is focused on how cells within developing fruit fly brain control the types, processing steps, and final sequences of the RNA molecules produced. We present data showing that when fly brain tissue lacks a protein called Nab2, some RNA molecules are not produced correctly. Nab2 loss causes extra sequences to be retained within many RNA molecules when those sequences are normally removed. These extra sequences can alter protein production from the affected RNAs and appear to contribute to the brain development problems observed in flies lacking Nab2. Since Nab2 performs very similar functions to a human protein called ZC3H14, these findings could provide a better understanding of how ZC3H14 loss leads to human disease.
Arsenic is a highly toxic metalloid that can have detrimental effects on farmed aquatic species, negatively impacting growth, metabolism, immunity and overall wellbeing of fish. The Indian major carp, Rohu (Labeo rohita) is a major freshwater aquaculture species that faces various production related issues associated with water quality parameters. The current study was designed to elucidate the effects of three different doses of arsenic (As) (T1 = 1 µg/L, T2 = 2 µg/L, and T3 = 3 µg/L) on the physiological (growth and O2 consumption), biochemical (blood cell counts), and genetic (expression levels of three selected genes) responses of Rohu. This study revealed significant (P<0.05) biologicl abnormalities and deformities in arsenic-exposed Rohu carp. The growth rate of fish decreased with increasing arsenic concentrations. O2 consumption rates of fish were increased (1.6-2 fold) with increasing experimental arsenic concentrations but blood cell counts were in a declining trend. Expression levels of the three selected genes showed arsenic dose specific differential changes; higher expression at control condition (1.5-2.1 fold) while lower expression at the treatment conditions. Results of this study clearly point out that different doses of arsenic impose stress at different orders of magnitude on the experimental Rohu individuals. The findings of the present study suggest that arsenic pollution significantly impacts the physiological (growth, development, metabolism and survivability), biochemical (hematological parameters) and molecular mechanisms of this economically important fish (L. rohita). Therefore, it is an imperative to maintain the optimum water quality (pollutant free) in the farming environments.
Pelvic organ prolapse (POP) afflicts millions of women globally. In POP, the weakened support of the pelvic floor results in the descent of pelvic organs into the vagina, causing a feeling of bulging, problems in urination, defaecation and/or sexual function. However, the existing surgical repair methods for relapsed POP remain insufficient, highlighting the urgent need for more effective alternatives. Collagen is an essential component in pelvic floor tissues, providing structural support, and its production is controlled by ascorbic acid. Therefore, we investigated novel ascorbic acid 2-phosphate (A2P)-releasing poly(l-lactide-co-ε-caprolactone) (PLCLA2P) membranes in vitro to promote cell proliferation and extracellular matrix protein production to strengthen the natural support of the pelvic fascia for POP applications. We analysed the mechanical properties and the impact of PLCLA2P on cellular responses through cell culture analysis using human vaginal fibroblasts (hVFs) and human adipose-derived stem/stromal cells (hASCs) compared to PLCL. In addition, the A2P release from PLCLA2P membranes was assessed in vitro. The PLCLA2P demonstrated slightly lower tensile strength (2.2 ± 0.4 MPa) compared to PLCL (3.7 ± 0.6 MPa) for the first 4 weeks in vitro. The A2P was most rapidly released during the first 48 h of in vitro incubation. Our findings demonstrated significantly increased proliferation and collagen production of both hVFs and hASCs on A2P-releasing PLCLA2P compared to PLCL. In addition, extracellular collagen Type I fibres were detected in hVFs, suggesting enhanced collagen maturation on PLCLA2P. Moreover, increased extracellular matrix protein expression was detected on PLCLA2P in both hVFs and hASCs compared to plain PLCL. In conclusion, these findings highlight the potential of PLCLA2P as a promising candidate for promoting tissue regeneration in applications aimed for POP tissue engineering applications.
Many insects and other animals carry microbial endosymbionts that influence their reproduction and fitness. These relationships only persist if endosymbionts are reliably transmitted from one host generation to the next. Wolbachia are maternally transmitted endosymbionts found in most insect species, but transmission rates can vary across environments. Maternal transmission of wMel Wolbachia depends on temperature in natural Drosophila melanogaster hosts and in transinfected Aedes aegypti, where wMel is used to block pathogens that cause human disease. In D. melanogaster, wMel transmission declines in the cold as Wolbachia become less abundant in host ovaries and at the posterior pole plasm (the site of germline formation) in mature oocytes. Here, we assess how temperature affects maternal transmission and underlying patterns of Wolbachia localization across 10 Wolbachia strains diverged up to 50 million years—including strains closely related to wMel—and their natural Drosophila hosts. Many Wolbachia maintain high transmission rates across temperatures, despite highly variable (and sometimes low) levels of Wolbachia in the ovaries and at the developing germline in late-stage oocytes. Identifying strains like closely related wMel-like Wolbachia with stable transmission across variable environmental conditions may improve the efficacy of Wolbachia-based biocontrol efforts as they expand into globally diverse environments.
Staphylococcus aureus and coagulase-negative staphylococci account for about 80% of infections associated with medical devices and are associated with increased virulence due to their ability to form biofilm. In this study, we aimed to construct a comprehensive reference map followed by significant pathway analysis in the proteome of S. aureus biofilm grown for 3-days as compared with 24 h planktonic using a high-resolution TMT based MS. We identified proteins associated with secondary metabolites, ABC transporters, biosynthesis of amino acids, response to stress, and amino sugar and nucleotide sugar metabolism were significantly upregulated in the 3-day biofilm. In contrast, proteins associated with virulence factors, microbial metabolism in diverse environments, translation, and energy metabolism were significantly downregulated. GO functional annotation indicated that more proteins are involved in metabolic processes, catalytic activity, and binding in biofilm, respectively. Among the significantly dysregulated proteins, hyaluronidase (hysA) in conjunction with chitinase may play a significant role in the elimination and/or prevention of biofilm development. This study advances the current S. aureus subproteomes, identified potential pathways significant to biofilm biology and helped to understand their potential role in S. aureus which may shed light on developing new therapeutic regimes including antibiofilm agents in the treatment of biofilm-infections related with implantable medical devices.
Staphylococcus aureus and coagulase-negative staphylococci account for about 80% of infections associated with medical devices and are associated with an increased virulence due to their ability to form biofilm. In this study, we aimed to construct a comprehensive reference map followed by significant pathway analysis in the proteome of S. aureus biofilm grown for 3-days as compared with 24 h planktonic using a high-resolution tandem mass tag (TMT)-based mass spectrometry. We identified proteins associated with secondary metabolites, ABC transporters, biosynthesis of amino acids, response to stress, and amino sugar and nucleotide sugar metabolism were significantly upregulated in 3-day biofilm. In contrast, proteins associated with virulence factors, microbial metabolism in diverse environments secondary metabolites, translation, and energy metabolism were significantly downregulated. Among the significantly dysregulated proteins, hyaluronidase (hysA) in conjunction with chitinase may play significant role in the elimination and/or prevention of biofilm development. This study advances the current S. aureus subproteomes, identified potential pathways significant to biofilm biology and helped to understand their potential role in S. aureus which may shed light on developing new therapeutic regimes including antibiofilm agents in the treatment of biofilm-infections related with implantable medical devices.
Background
Sleep disturbance and fatigue are common in individuals undergoing inpatient rehabilitation following stroke. Understanding the relationships between sleep, fatigue, motor performance, and key biomarkers of inflammation and neuroplasticity could provide valuable insight into stroke recovery, possibly leading to personalized rehabilitation strategies. This study aimed to investigate the influence of sleep quality on motor function following stroke utilizing wearable technology to obtain objective sleep measurements. Additionally, we aimed to determine if there were relationships between sleep, fatigue, and motor function. Lastly, the study aimed to determine if salivary biomarkers of stress, inflammation, and neuroplasticity were associated with motor function or fatigue post-stroke.
Methods
Eighteen individuals who experienced a stroke and were undergoing inpatient rehabilitation participated in a cross-sectional observational study. Following consent, participants completed questionnaires to assess sleep patterns, fatigue, and quality of life. Objective sleep was measured throughout one night using the wearable Philips Actiwatch. Upper limb motor performance was assessed on the following day and saliva was collected for biomarker analysis. Correlation analyses were performed to assess the relationships between variables.
Results
Participants reported poor sleep quality, frequent awakenings, and difficulties falling asleep following stroke. We identified a significant negative relationship between fatigue severity and both sleep quality (r=-0.539, p = 0.021) and participants experience of awakening from sleep (r=-0.656, p = 0.003). A significant positive relationship was found between grip strength on the non-hemiplegic limb and salivary gene expression of Brain-derived Neurotrophic Factor (r = 0.606, p = 0.028), as well as a significant negative relationship between grip strength on the hemiplegic side and salivary gene expression of C-reactive Protein (r=-0.556, p = 0.048).
Conclusion
The findings of this study emphasize the importance of considering sleep quality, fatigue, and biomarkers in stroke rehabilitation to optimize recovery and that interventions may need to be tailored to the individual. Future longitudinal studies are required to explore these relationships over time. Integrating wearable technology for sleep and biomarker analysis can enhance monitoring and prediction of outcomes following stroke, ultimately improving rehabilitation strategies and patient outcomes.
The protozoan parasite Leishmania spp. is a causative agent of leishmaniasis, a disease that affects millions of people in more than 80 countries worldwide. Apart from its medical relevance, this organism has a genetic organization that is unique among eukaryotes. Studies of the mechanisms regulating gene expression in Leishmania led us to investigate noncoding RNAs (ncRNAs) as regulatory elements. We previously identified differentially expressed (DE) ncRNAs in Leishmania braziliensis with potential roles in the parasite biology and development. Herein, we present a functional analysis of one such DE ncRNA, the 147-nucleotide-long transcript ncRNA97, which is preferentially expressed in amastigotes, the replicative form within mammalian phagocytes. By RT-qPCR the ncRNA97 was detected in greater quantities in the nucleus under physiological conditions and in the cytoplasm under nutritional stress. Interestingly, the transcript is protected at the 5' end but is not processed by the canonical trypanosomatid trans-splicing mechanism, according to the RNA circularization assay. ncRNA97 knockout (KO) and addback (AB) transfectants were generated and subjected to phenotypic analysis, which revealed that ncRNA97 impairs the starvation response and differentiation to the infective form. Comparative transcriptomics of ncRNA97KO and parental cells revealed that transcripts encoding amastigote-specific proteins were affected. This pioneering work demonstrates that ncRNAs contribute to the developmental regulatory mechanisms of Leishmania .
The transcription factor GT2‐LIKE 1 (GTL1) has been implicated in orchestrating a transcriptional network of diverse physiological, biochemical, and developmental processes. In response to water‐limiting conditions, GTL1 is a negative regulator of stomatal development, but its potential rolein other water‐deficit responses is unknown. We hypothesized that GTL1 regulates transcriptome changes associated with drought tolerance over leaf developmental stages. To test the hypothesis, gene expression was profiled by RNA‐seq analysis in emerging and expanding leaves of wild‐type and a drought‐tolerant gtl1‐4 knockout mutant under well‐watered and water‐deficit conditions. Our comparative analysis of genotype‐treatment combinations within leaf developmental age identified 459 and 1073 differentially expressed genes in emerging and expanding leaves, respectively, as water‐deficit responsive GTL1‐regulated genes. Transcriptional profiling identified a potential role of GTL1 in two important pathways previously linked to drought tolerance: flavonoid and polyamine biosynthesis. In expanding leaves, negative regulation of GTL1 under water‐deficit conditions promotes biosynthesis of flavonoids and anthocyanins that may contribute to drought tolerance. Quantification of polyamines did not support a role for GTL1 in these drought‐responsive pathways, but this is likely due to the complex nature of polyamine synthesis and turnover. Our global transcriptome analysis suggests that transcriptional repression of GTL1 by water deficit allows plants to activate diverse pathways that collectively contribute to drought tolerance.
The gut microbiota contributes to skeletal muscle energy metabolism and is an indirect factor affecting meat quality. However, the role of specific gut microbes in energy metabolism and fiber size of skeletal muscle in chickens remains largely unknown. In this study, we first performed cecal microbiota transplantation from Chinese indigenous Jingyuan chickens (JY) to Arbor Acres chickens (AA), to determine the effects of microbiota on skeletal muscle fiber and energy metabolism. Then, we used metagenomics, gas chromatography, and metabolomics analysis to identify functional microbes. Finally, we validated the role of these functional microbes in regulating the fiber size via glucose metabolism in the skeletal muscle of chickens through feeding experiments. The results showed that the skeletal muscle characteristics of AA after microbiota transplantation tended to be consistent with that of JY, as the fiber diameter was significantly increased, and glucose metabolism level was significantly enhanced in the pectoralis muscle. L. plantarum, L. ingluviei, L. salivarius, and their mixture could increase the production of the microbial metabolites protoporphyrin IX and short-chain fatty acids, therefore increasing the expression levels of genes related to the oxidative fiber type (MyHC SM and MyHC FRM), mitochondrial function (Tfam and CoxVa), and glucose metabolism (PFK, PK, PDH, IDH, and SDH), thereby increasing the fiber diameter and density. These three Lactobacillus species could be promising probiotics to improve the meat quality of chicken.
IMPORTANCE
This study revealed that the L. plantarum, L. ingluviei, and L. salivarius could enhance the production of protoporphyrin IX and short-chain fatty acids in the cecum of chickens, improving glucose metabolism, and finally cause the increase in fiber diameter and density of skeletal muscle. These three microbes could be potential probiotic candidates to regulate glucose metabolism in skeletal muscle to improve the meat quality of chicken in broiler production.
Statins, the drugs used for the treatment of hypercholesterolemia, have come into the spotlight not only as chemoadjuvants, but also as potential stem cell modulators in the context of regenerative therapy. In our study, we compared the in vitro effects of all clinically used statins on the viability of human pancreatic cancer (MiaPaCa-2) cells, non-cancerous human embryonic kidney (HEK 293) cells and adipose-derived mesenchymal stem cells (ADMSC). Additionally, the effect of statins on viability of MiaPaCa-2 and ADMSC cells spheroids was tested. Furthermore, we performed a microarray analysis on ADMSCs treated with individual statins (12 μM) and compared the importance of the effects of statins on gene expression between stem cells and pancreatic cancer cells. Concentrations of statins that significantly affected cancer cells viability (< 40 μM) did not affect stem cells viability after 24 h. Moreover, statins that didn´t affect viability of cancer cells grown in a monolayer, induce the disintegration of cancer cell spheroids. The effect of statins on gene expression was significantly less pronounced in stem cells compared to pancreatic cancer cells. In conclusion, the low efficacy of statins on non-tumor and stem cells at concentrations sufficient for cancer cells growth inhibition, support their applicability in chemoadjuvant tumor therapy.
Colorectal cancer (CRC) arises via the progressive accumulation of dysregulation in key genes including oncogenes and tumor-suppressor genes. Prostaglandin-endoperoxide synthase 2 (PTGS2, also called COX2) acts as an oncogenic driver in CRC. Here, we explored the upstream transcription factors (TFs) responsible for elevating PTGS2 expression in CRC cells. The results showed that PTGS2 silencing repressed cell growth, migration and invasion in HCT116 and SW480 CRC cells. The two fragments (499–981 bp) and (1053–1434 bp) were confirmed as the core TF binding profiles of the PTGS2 promoter. PTGS2 expression positively correlated with RUNX1 level in colon adenocarcinoma (COAD) samples using the TCGA-COAD dataset. Furthermore, RUNX1 acted as a positive regulator of PTGS2 expression by promoting transcriptional activation of the PTGS2 promoter via the 1086–1096 bp binding motif. In conclusion, our study demonstrates that PTGS2 upregulation induced by the TF RUNX1 promotes CRC cell growth, migration and invasion, providing an increased rationale for the use of PTGS2 inhibitors in CRC prevention and treatment.
Continuous fluorescence observation of amplifying DNA allows rapid and accurate quantification of initial transcript copy number A simple and generic method for monitoring product synthesis with the double-stranded DNA dye, SYBR(R) Green I provides initial template copy number estimation limited only by stochastic effects. To reach this degree of sensitivity, two methods were used. First, specific products generally have a higher melting temperature than nonspecific products, and therefore, specific product formation was monitored by fluorescence acquisition at temperatures at which only specific products are double-stranded. Second, anti-Tag antibodies were used to reduce nonspecific product generation. The log-linear portion of the fluorescence vs. cycle plot was extended to determine a fractional cycle number at which a threshold fluorescence was obtained These fractional cycle numbers were plotted against the log of starting template copies to give linear standard curves from purified PCR products, allowing easy estimation of cDNA unknowns over a 10(6)-fold range. A single template molecule per reaction could be distinguished from the absence of template, although stochastic effects increased the variance of concentration estimates below 10 copies. Above 10 copies per reaction, typical replicate coefficients of variation were 6%-37%, with better precision at higher copy numbers.
The efficiency of three commonly used reverse transcriptases, namely Moloney Murine Leukaemia Virus (M-MuLV) reverse transcriptase, RNase H--M-MuLV reverse transcriptase and Avian Myeloblastoma Leukaemia Virus (AMV) reverse transcriptase (RT), have been compared in RT-PCR amplification of antibody genes. These enzymes differed significantly in their efficiency of cDNA synthesis as indicated by the degree of V gene amplification by PCR; Moloney Murine Leukaemia Virus (M-MuLV) reverse transcriptase and RNase H--M-MuLV reverse transcriptase produced greater amplification of the human V-gene repertoire using RT-PCR than that of the Avian Myeloblastoma Leukaemia Virus (AMV) reverse transcriptase.
We describe a simple, quantitative assay for any amplifiable DNA sequence that uses a video camera to monitor multiple polymerase chain reactions (PCRs) simultaneously over the course of thermocycling. The video camera detects the accumulation of double-stranded DNA (dsDNA) in each PCR using the increase in the fluorescence of ethidium bromide (EtBr) that results from its binding duplex DNA. The kinetics of fluorescence accumulation during thermocycling are directly related to the starting number of DNA copies. The fewer cycles necessary to produce a detectable fluorescence, the greater the number of target sequences. Results obtained with this approach indicate that a kinetic approach to PCR analysis can quantitate DNA sensitively, selectively and over a large dynamic range. This approach also provides a means of determining the effect of different reaction conditions on the efficacy of the amplification and so can provide insight into fundamental PCR processes.
In this paper we determine the limits and accuracy of quantitative reverse transcription (RT)-PCR using a modification of the original protocol. The quantification of mRNA with this procedure requires a preliminary estimation of the target molecule (TM) concentration, established from experiments with an internal control molecule (ICM). A definitive quantification is then attained from serial dilutions of the reverse transcription reaction. The success of this latter step is dependent on maintaining an equivalent number of TM and ICM in the reaction. The purpose of our study was to evaluate the influence of the deviation between the TM and the ICM on the result. We show here that we can control the accuracy of the assay by fixing the limit of the TM/ICM ratio. Indeed, when the TM/ICM ratio is between 0.66 and 1.5 (i.e., the difference between TM and ICM is 1.5-fold), the final result has an error of approximately 10%. Exceeding this limit produces errors approaching 60%, as in the case of TM/ICM = 2. When the above conditions are respected, a difference as small as 20% between two samples can be determined with an accuracy of 95%.
A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR) target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the CFTR amplicon. Relative increase in 6-carboxy-fluorescein reporter fluorescent emission is monitored during PCR amplification using an analytical thermal cycler. An internal control template containing the same primer sequences as the CFTR amplicon, but a different internal sequence, has been designed as a control. An internal control probe with a reporter fluorescent dye tetrachloro-6-carboxy-fluorescein was designed to hybridize to the internal control amplicon. The internal control template is placed in each reaction tube and is used for quantitative analysis of the CFTR mRNA. This method provides a convenient and high-throughput format for QC RT-PCR.
Experimental and commercial microvolume fluorimeters with rapid temperature control are described. Fluorescence optics adopted from flow cytometry were used to interrogate 1-10-microL samples in glass capillaries. Homogeneous temperature control and rapid change of sample temperatures (10 degrees C/s) were obtained by a circulating air vortex. A prototype 2-color, 32-sample version was constructed with a xenon arc for excitation, separate excitation and emission paths, and photomultiplier tubes for detection. The commercial LightCycler, a 3-color, 24-sample instrument, uses a blue light-emitting diode for excitation, paraxial epi-illumination through the capillary tip and photodiodes for detection. Applications include analyte quantification and nucleic acid melting curves with fluorescent dyes, enzyme assays with fluorescent substrates and techniques that use fluorescence resonance energy transfer. Microvolume capability allows analysis of very small or expensive samples. As an example of one application, rapid cycle DNA amplification was continuously monitored by three different fluorescence techniques, Which included using the double-stranded DNA dye SYBR Green I, a dual-labeled 5'-exonuclease hydrolysis probe, and adjacent fluorescein and Cy5z-labeled hybridization probes. Complete amplification and analysis requires only 10-15 min.
Various "housekeeping" genes are often used as endogenous controls in gene expression experiments. We have cloned from swine, three genes commonly used as endogenous controls in other species and have characterized their relative levels of expression in various porcine tissues and their response to various cell activators. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin were readily detected by northern hybridization in various tissues and in alveolar macrophages. The expression of hypoxanthine phosphoribosyltransferase (HPRT) was detected only by northern hybridization of poly-A+ enriched RNA and by reverse transcriptase-polymerase chain reaction (RT-PCR), making it more suitable for highly sensitive detection methods. Expression of GAPDH varied less among tissues than did beta-actin, making it more useful control for comparisons of gene expression between tissues with northern hybridizations. Various treatments of cultured alveolar macrophages differentially affected levels of beta-actin and GAPDH, while HPRT expression was unchanged in alveolar macrophages or spleen cells similarly treated. Therefore, while HPRT can be used as the endogenous control with sensitive detection methods such as RT-PCR, less sensitive detection methods require a more abundant gene such as GAPDH.
Reverse transcription PCR (RT-PCR) represents a sensitive and powerful tool for analyzing RNA. While it has tremendous potential for quantitative applications, a comprehensive knowledge of its technical aspects is required. Successful quantitative RT-PCR involves correction for experimental variations in individual RT and PCR efficiencies. This review addresses the mathematics of RT-PCR, choice of RNA standards (internal vs. external) and quantification strategies (competitive, noncompetitive and kinetic [real-time] amplification). Finally, the discussion turns to practical considerations in experimental design. It is hoped that this review will be appropriate for those undertaking these experiments for the first time or wishing to improve (or validate) a technique in what is frequently a confusing and contradictory field.
The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).
Internally standardised competitive RT-PCR measured by HPLC separation and UV detection [9] or high resolution gel electrophoresis followed by densitometric analysis [8]: In a competitive RT-PCR, a reference RNA mutant is reverse transcribed and co-amplified in the same reaction tube with the native mRNA sequence of interest. Internally standardised RT-PCR is a very time-consuming and laborious technique. It is generally believed to yield the most precise results, because all parameters throughout RT-PCR act on both the analyte and reference mutant.
Over the last 15 years, PCR has become an essential part of most laboratories involved in biomedical research. PCR amplification turns a few attograms of a specific fragment of nucleic acid (far too little to be analyzed directly or used in biochemical reactions) into as much as a microgram of DNA.
Reverse transcription (RT) followed by polymerase chain reaction (PCR) is the technique of choice for analysing mRNA in extremely low abundance. Real-time RT-PCR using SYBR Green I detection combines the ease and necessary exactness to be able to produce reliable as well as rapid results. To obtain high accuracy and reliability in RT and real-time PCR a highly defined calibration curve is needed. We have developed, optimised and validated an Insulin-like growth factor-1 (IGF-1) RT-PCR in the LightCycler, based on either a recombinant IGF-1 RNA (recRNA) or a recombinant IGF-1 DNA (recDNA) calibration curve. Above that, the limits, accuracy and variation of these externally standardised quantification systems were determined and compared with a native RT-PCR from liver total RNA. For the evaluation and optimisation of cDNA synthesis rate of recRNA several RNA backgrounds were tested. We conclude that external calibration curve using recDNA is a better model for the quantification of mRNA than the recRNA calibration model. This model showed higher sensitivity, exhibit a larger quantification range, had a higher reproducibility, and is more stable than the recRNA calibration curve.
Quantitative studies are commonly realised in the biomedical research to compare RNA expression in different experimental or clinical conditions. These quantifications are performed through their comparison to the expression of the housekeeping gene transcripts like glyceraldehyde-3-phosphate dehydrogenase (G3PDH), albumin, actins, tubulins, cyclophilin, hypoxantine phsophoribosyltransferase (HRPT), L32. 28S, and 18S rRNAs are also used as internal standards. In this paper, it is recalled that the commonly used internal standards can quantitatively vary in response to various factors. Possible variations are illustrated using three experimental examples. Preferred types of internal standards are then proposed for each of these samples and thereafter the general procedure concerning the choice of an internal standard and the way to manage its uses are discussed.
Nitric oxide generation in brain cytosolic fractions markedly enhances ADP-ribosylation of a single 37-kDa protein. By utilizing a biotinylated NAD and avidin affinity chromatography, we purified this protein. Partial amino acid sequencing establishes its identity as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This is further confirmed by detection of GAPDH enzymatic activity in the purified 37-kDa protein. GAPDH is ADP-ribosylated in the absence of brain extract. This auto-ADP-ribosylation is enhanced by nitric oxide generation. ADP-ribosylation appears to involve the cysteine where NAD interacts with GAPDH so that ADP-ribosylation likely inhibits enzymatic activity. Such inhibition may play a role in nitric oxide-mediated neurotoxicity.
A method for the quantitation of specific mRNA species by the polymerase chain reaction (PCR) has been developed by using a synthetic RNA as an internal standard. The specific target mRNA and the internal standard are coamplified in one reaction in which the same primers are used. The amount of mRNA is then quantitated by extrapolating against the standard curve generated with the internal standard. The synthetic internal standard RNA consists of a linear array of the sequences of upstream primers of multiple target genes followed by the complementary sequences to their downstream primers in the same order. This quantitative PCR method provides a rapid and reliable way to quantify the amount of a specific mRNA in a sample of less than 0.1 ng of total RNA. In addition, the same internal standard RNA is used, with appropriate primer pairs, to quantitate multiple different mRNA species.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been a gene of choice in Northern blot analyses as an internal RNA loading control. We have investigated the expression of GAPDH and 28S-ribosomal RNA (28S rRNA) genes in mouse 10T1/2 cells and in a variety of tumorigenic and highly malignant metastatic cell lines derived from the 10T1/2 cell line. We observed that GAPDH mRNA levels varied markedly among the tumorigenic and highly malignant cell lines and were elevated in these cell lines when compared to the normal mouse 10T1/2 cells. In contrast, the levels of 28S rRNA did not significantly vary among the tumorigenic and highly malignant cell lines and were approximately at the same level as that found in the normal parental mouse 10T1/2 cell line. These observations indicate that much caution should be taken when using GAPDH gene expression as an RNA loading control for Northern blots. Based upon these observations, we recommend the use of 28S rRNA gene expression as a preferred RNA loading control for Northern blot analysis in which total RNA is used.
We showed previously that the abundance of serum albumin mRNA is decreased in H4-II-E rat hepatoma cells limited for a single essential amino acid (phenylalanine, methionine, leucine, or tryptophan). To define the specificity of this phenomenon, we examined the effect of amino acid limitation on the abundance of mRNAs for 19 genes in the H4-II-E cells. These genes included six genes whose expression is either completely liver-specific or highly enriched in the liver compared with other tissues [albumin, transthyretin (TTR), transferrin, carbamyl phosphate synthetase-I, urate oxidase, class I alcohol dehydrogenase], as well as a number of ubiquitously expressed "housekeeping" genes. The results indicated that the 19 genes could be divided into three classes based on their response to amino acid limitation. Class I genes (the six liver-specific genes and alpha-tubulin) exhibit decreased expression in response to amino acid limitation. The expression of class II genes [beta 2-microglobulin, hypoxanthine-guanine phosphoribosyl transferase (HPRT), H-ferritin, ubiquitin (UbB), insulin-like growth factor binding protein-4, HNF-1 alpha] is not significantly affected by amino acid limitation. Class III genes [gadd153, beta-actin, ubiquitin (UbC), phosphoglycerate kinase-1, C/EBP alpha, C/EBP beta] exhibit increased expression in response to amino acid limitation. Thus, specific inductive as well as repressive effects on gene expression are quite common in amino acid-limited cells. The observation that all six genes whose expression is liver-specific exhibited decreased expression in amino acid-limited cells suggests a common mode of regulation of these genes by amino acid availability. The strong induction by amino acid limitation of the C/EBP inhibitor gadd153 is of interest in this regard, as increased levels of gadd153 could interfere with C/EBP, which is required for high expression of most liver-specific genes. To investigate further the molecular mechanism for the decrease in albumin mRNA abundance, albumin nuclear transcript levels were quantified in control and tryptophan-limited cells. Tryptophan limitation caused a decrease in albumin nuclear transcript abundance, and this decrease preceded the decrease in albumin mRNA, suggesting that the decrease in albumin mRNA was caused at least partly by a decrease in albumin gene transcription. Additional experiments with actinomycin D indicated that albumin mRNA was also destabilized in the tryptophan-limited cells. Thus, the overall results indicate that the decrease in albumin mRNA in the tryptophan-limited cells is caused by a specific decrease in albumin nuclear transcript abundance and destabilization of albumin mRNA.
Previous studies have shown that endogenous nitric oxide (NO) potentiates glycolysis in the cytokine-activated murine microvascular endothelial cells (MME). In the present study we investigate the influence of NO on the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme of the glycolytic pathway. Activation of MME with TNF-alpha and IFN-gamma resulted in a strong elevation of GAPDH mRNA levels. This effect was impaired in the presence of L-NMMA, the inhibitor of NO synthesis. We discuss the possibility that NO-mediated elevation of GAPDH mRNA levels may compensate for NO-mediated inhibition of GAPDH enzymatic activity, representing another adaptive mechanism which protects cells producing large amounts of NO against its cytotoxic effects.
Beta-actin, cyclophilin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are all constantly expressed proteins that regulate cellular structures and endogenous cytoarchitectural functions. In this study, we used an in vivo N1S1 rat hepatoma model to examine changes in the expression levels of these housekeeping genes in normal and tumor liver samples. The beta-actin, cyclophilin and GAPDH genes were all up-regulated in tumor groups as compared to the controls. Our results suggest that up-regulation of beta-actin, cyclophilin and GAPDH genes may be essential for oncogenesis in hepatoma.
In recent years the growing interest in quantitative applications of the polymerase chain reaction (PCR) has favoured the development of a large number of assay procedures suitable for this purpose. In this paper we review some basic principles of quantitative PCR and in particular the role of reference materials and calibrators and the different strategies adopted for nucleic acid quantification. We focus on two methodological approaches for quantitative PCR in this review: competitive PCR and real-time quantitative PCR based on the use of fluorogenic probes. The first is one of the most common methods of quantitative PCR and we discuss the structure of the competitors and the various assay procedures. The second section is dedicated to a recent promising technology for quantitative PCR in which the use of fluorogenic probes and dedicated instrumentation allows the development of homogeneous methods. Assay performance of these methods in terms of practicability and reliability indicates that these kinds of technologies will have a widespread use in the clinical laboratory in the near future.
To investigate the role of local IGF-1 mRNA expression in various tissues, we developed and validated a method which allows for a specific, sensitive and reliable quantification of IGF-1 mRNA: an internally standardised Reverse Transcription-Polymerase Chain Reaction (RT-PCR). A synthetic competitive template IGF-I standard cRNA (IGF-1 cRNA) was designed, which contains the same flanking primer sequences used to amplify the wild type IGF-1 mRNA, but differs by 56 bp in length. To obtain the IGF-1 mRNA concentration present in tissue RNA samples, series of 250 ng total-RNA were spiked with three known quantities of the standard IGF-1 cRNA, incubated for competitive RT-PCR reactions and the two amplificates obtained (184 bp from IGF-1 cRNA and 240 bp from the wild type IGF-I mRNA) were subsequently separated and quantified by HPLC-UV. For every individual tissue RNA sample, the ratio R (R = competitor PCR product / wild type PCR product) was plotted against the number of starting molecules of the competitor IGF-1 cRNA. The initial amount of IGF-1 mRNA present in the sample can then be read off where R = 1. The validated assay had a detection limit of 1600 IGF-1 cRNA molecules/reaction, the intra-assay variation was 7.4% (n = 5) and linearity (r = 0.997) was given between 140 ng to 840 ng total-RNA input. The present method was first applied to study the effect of long term castration on the IGF-1 expression rates in bovine tissues. The hepatic IGF-1 mRNA concentrations were well correlated (r = 0.81) with the plasma concentrations as quantified by RIA and were higher in intact than in castrated animals. In two skeletal muscles (m. splenius and m. gastrocnemius) IGF-1 mRNA concentrations were 20- and 35- times lower than in liver, respectively, without any differences between steers and bulls. In bulls, the IGF-1 mRNA expression was higher in m. splenius (p < 0.01) than m. gastrocnemius, indicating that locally produced IGF-1 might be important for sexually dimorphic muscle growth patterns.
Recently, a novel technique for "real time" quantitative Reverse Transcriptase-PCR which measures PCR-product accumulation during the exponential phase of the PCR reaction using a dual-labelled fluorogenic probe, has been developed. This method allows direct detection of PCR-product formation by measuring the increase in fluorescent emission continuously during the PCR reaction. Here we present data validating this PCR-method for the quantification of murine cytokines and other factors playing a role in immune regulation (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-13, IL-15, IFN-gammaTNF-alphaTGF-beta and iNOS). For each substance of interest, a set of primers and internal probe was designed, which specifically amplify the target cDNA, not co-amplifying contaminating genomic DNA. Furthermore, a corresponding reference plasmid cDNA clone was constructed, allowing direct quantification. Additionally, normalization to the housekeeping genes beta-actin or GAPDH was performed. The assay is very sensitive and accurate. It is a "closed-tube" PCR reaction, avoiding time-consuming and hazardous post-PCR manipulations and decreasing the potential risk of PCR contamination.
Reverse transcription polymerase chain reaction (RT-PCR) analysis is increasingly becoming part of the diagnostic and prognostic evaluation for hematologic and oncologic disorders. Currently, different RNA isolation methods are used in the diagnostic laboratories. No data are available on their suitability for sensitive detection of breakpoint cluster region-abelson (BCR-ABL) gene transcripts. We have extracted RNA from mononuclear cell (MNC) fractions and from lysed blood samples of 4 patients (1 with leukocytosis, 1 with chronic myelogeneous leukemia (CML) under interferon treatment, and 2 CML patients after bone marrow transplantation) with 3 RNA isolation reagents (TRIzol, RNAzol, FastTube reagent). RNA yield was slightly higher with RNAzol than with TRIzol as indicated by agarose gel electrophoresis and spectrophotometric measurement at 260 nm. The FastTube reagent was unsuitable for RNA isolation from MNC, and was not evaluated for lysed blood. Quantitative competitive RT-PCR amplification of the ABL gene showed comparable results for RNA isolated with RNAzol and TRIzol. In RNA samples extracted from lysed whole blood, the presence of amplifiable RNA/cDNA was confirmed by amplification of 4 selected reference genes (porphobilinogen deaminase (PBGD), ABL, the gene spanning the BCR on chromosome 22 and retinoic acid receptor alpha (RARA)) in a multiplex PCR. High quality, DNA-free RNA was obtained with RNAzol, and 1 BCR-ABL-positive (specific for translocation t [9; 221) cell among 2x10(4) normal cells was successfully detectable by single step RT-PCR. In RNA isolated with TRIzol, major contaminations with genomic DNA were observed which significantly impaired the interpretation of the results of RT-PCR analysis.
Detection of colorectal micrometastasis by quantitative RT-PCR of cytokeratin 20 mRNA
- R Soong
- J Ruschoff
- K Tabiti
Soong,R., Ruschoff,J. and Tabiti,K. (2000) Detection of colorectal
micrometastasis by quantitative RT-PCR of cytokeratin 20 mRNA.
Roche Diagnostics internal publication.