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Summary: Real-time RT PCR has been recognized as an
accurate, reliable and sensitive method for quantifying gene
transcription. However, several steps preceding PCR represent
critical points and source of inaccuracies. These steps
include cell processing, RNA extraction, RNA storage,
assessment of RNA concentration and cDNA synthesis. To
compensate...
Contexts in source publication
Context 1
... selectivity of PCR results is achieved in this step by using the primers comple- mentary to the sequences outlining the targeted DNA region. During the elongation step, DNA polymerase creates two double strand target regions, each of which can again be denatured and ready for a second cycle of annealing (hybridization) and elongation ( Figure 1). ...
Context 2
... researchers, the MIQE checklist represents a good starting point. It helps investigators to wisely plan and conduct experiments, as well as to clearly write and present the results (Figure 7) (90, 91). Namely, it is important to provide complete technical information in a manuscript, to validate protocols and Figure 6 Relative expression of gap43 measured 6, 12 and 18 months after a treatment and normalized to refer- ence gene 1 and reference gene 2. ...
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The selection and validation of stably expressed reference genes is a critical issue for proper RT-qPCR data normalization. In zebrafish expression studies, many commonly used reference genes are not generally applicable given their variability in expression levels under a variety of experimental conditions. Inappropriate use of these reference gen...
Background
Quantitative real-time reverse-transcription polymerase chain reaction is frequently used as research tool in experimental oncology. There are some studies of valid endogenous control genes in the field of human glioma research, which, however, only focus on the comparison between normal brain with tumor tissue and malignant transformati...
Citations
... Given the variability in gene expression across diverse experimental settings, it is advisable to thoroughly validate candidate reference genes tailored to the specific experimental conditions prior to their utilization for qPCR normalization. Relying solely on reference genes from previous studies conducted under differing experimental conditions may not ensure precise normalization and could lead to inaccuracies in the analysis [43]. In crustaceans, the EF-1α, GAPDH, 18S rRNA, RpL8, RpL18, and β-actin genes are commonly employed as internal controls across a range of experimental conditions [14,17]. ...
Background
Gene expression profiling via qPCR is an essential tool for unraveling the intricate molecular mechanisms underlying growth and development. Identifying and validating the most appropriate reference genes is essential for qPCR experiments. Nevertheless, there exists a deficiency in a thorough assessment of reference genes concerning the expression of the genes in the research in the context of the growth and development of the Black Tiger Shrimp, P. monodon. This popular marine crustacean is extensively raised for human consumption. In this study, we assessed the expression stability of seven reference genes (ACTB, 18S, EF-1α, AK, PK, cox1, and CLTC) in adult tissues (hepatopancreas, gills, and stomach) of small and large polymorphs of P. monodon.
Methods and results
The stability of gene expressions was assessed utilizing NormFinder, BestKeeper, and geNorm, and a comprehensive ranking of these genes was conducted through the online tool RefFinder. In the overall ranking, 18S and CLTC emerged as the most stable genes in the hepatopancreas and stomach, while CLTC and AK exhibited significant statistical reliability in the gills of adult P. monodon. The validation of these identified stable genes was carried out using a growth-associated gene, insr-1.
Conclusion
The results indicated that 18S and CLTC stand out as the most versatile reference genes for conducting qPCR analysis focused on the growth of P. monodon. This study represents the first comprehensive exploration that identifies and assesses reference genes for qPCR analysis in P. monodon, providing valuable tools for research involving similar crustaceans.
... However, some factors can easily affect the method's accuracy and precision. The reliability of the obtained results largely depends on the quality and quantity of mRNA used as a template for the qRT-PCR reaction, as well as on factors such as the efficiency of the reverse transcription reaction, primer design and concentration, incubation time, running temperature, annealing temperature, template concentration, and reaction conditions [2,3]. Nevertheless, even if all steps are performed correctly, a crucial aspect is crucial to validate the expression stability of candidate reference genes under specific trial conditions before using them in gene expression analyses. ...
Obtaining accurate and reliable gene expression results in real-time RT-PCR (qRT-PCR) data analysis requires appropriate normalization by carefully selected reference genes, either a single or a combination of multiple housekeeping genes (HKGs). The optimal reference gene/s for normalization should demonstrate stable expression across varying conditions to diminish potential influences on the results. Despite the extensive database available, research data are lacking regarding the most appropriate HKGs for qRT-PCR data analysis in rabbit and horse adipose-derived stem cells (ASCs). Therefore, in our study, we comprehensively assessed and compared the suitability of some widely used HKGs, employing RefFinder and NormFinder, two extensively acknowledged algorithms for robust data interpretation. The rabbit and horse ASCs were obtained from subcutaneous stromal vascular fraction. ASCs were induced into tri-lineage differentiation, followed by the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) treatment of the adipose-differentiated rabbit ASCs, while horse experimental groups were formed based on adipogenic, osteogenic, and chondrogenic differentiation. At the end of the experiment, the total mRNA was obtained and used for the gene expression evaluation of the observed factors. According to our findings, glyceraldehyde 3-phosphate dehydrogenase was identified as the most appropriate endogenous control gene for rabbit ASCs, while hypoxanthine phosphoribosyltransferase was deemed most suitable for horse ASCs. The obtained results underscore that these housekeeping genes exhibit robust stability across diverse experimental conditions, remaining unaltered by the treatments. In conclusion, the current research can serve as a valuable baseline reference for experiments evaluating gene expression in rabbit and horse ASCs. It highlights the critical consideration of housekeeping gene abundance and stability in qPCR experiments, emphasizing the need for an individualized approach tailored to the specific requirements of the study.
... The polymerase chain reaction (PCR) was developed in 1983 by Mullis [36]. This method quickly contributed to significant progress in many fields of science and in diagnostics. ...
... Real-time PCR has been widely used for the detection and differentiation of microorganisms from various samples, both environmental and clinical, since the method allows amplification of a specific fragment of DNA [36]. For example, based on qPCR, Trung and colleagues developed a method for direct detection and quantification of Burkholderia pseudomallei. ...
The increasing number of patients with chronic wounds requires the development of quick and accurate diagnostics methods. One of the key and challenging aspects of treating ulcers is to control wound infection. Early detection of infection is essential for the application of suitable treatment methods, such as systemic antibiotics or other antimicrobial agents. Clinically, the most frequently used method for detecting microorganisms in wounds is through a swab and culture on appropriate media. This test has major limitations, such as the long bacterial growth time and the selectivity of bacterial growth. This article presents an overview of molecular methods for detecting bacteria in wounds, including real-time polymerase chain reaction (rtPCR), quantitative polymerase chain reaction (qPCR), genotyping, next-generation sequencing (NGS), and loop-mediated isothermal amplification (LAMP). We focus on the LAMP method, which has not yet been widely used to detect bacteria in wounds, but it is an interesting alternative to conventional detection methods. LAMP does not require additional complicated equipment and provides the fastest detection time for microorganisms (approx. 30 min reaction). It also allows the use of many pairs of primers in one reaction and determination of up to 15 organisms in one sample. Isothermal amplification of DNA is currently the easiest and most economical method for microbial detection in wound infection. Direct visualization of the reaction with dyes, along with omitting DNA isolation, has increased the potential use of this method.
... The practice of incorporating multiple reference genes to standardize the levels of target-gene expression has been widely accepted due to its ability to mitigate diverse errors and guarantee the precision of experimental outcomes [54,55]. Nevertheless, an inadequate or unreliable reference gene may result in an erroneous depiction of the target gene's expression pattern, consequently leading to flawed interpretations [56,57]. The OBP gene plays a crucial role in the initial recognition processes of semiochemical perception and frequently exhibits expression patterns specific to certain tissues [58,59]. ...
Quantitative real-time PCR (qRT-PCR) is widely accepted as a precise and convenient method for quantitatively analyzing the expression of functional genes. The data normalization strongly depends upon stable reference genes. The bean bug, Riptortus pedestris (Hemiptera: Alydidae), is a significant pest of leguminous crops and broadly distributed across Southeast Asia. In this study, a total of 16 candidate reference genes (RPL32, RPS23, SDHA, UBQ, UCCR, GST, TATA−box, HSP70, GAPDH, RPL7A, SOD, RPS3, Actin, α−tubulin, AK, and EF1) were carefully chosen in R. pedestris, and their expression levels were assessed across various conditions, including different developmental stages, diverse tissues, temperature treatments, adult age, molting time, and mating status. Following this, the stability of these reference genes was evaluated using four algorithms (ΔCt, GeNorm, NormFinder, and BestKeeper). Ultimately, the comprehensive rankings were determined using the online tool RefFinder. Our results demonstrate that the reference gene for qRT-PCR analysis in R. pedestris is contingent upon the specific experimental conditions. RPL7A and EF1 are optimal reference genes for developmental stages. Furthermore, α−tubulin and EF1 exhibit the most stable expression across various adult tissues. RPL32 and RPL7A exhibit the most stable expression for adult age. For nymph age, RPL32 and SOD display the most stable expression. For temperature conditions, RPS23 and RPL7A were identified as the most suitable for monitoring gene expression. Lastly, we verified the practicability of evaluating expression levels of odorant-binding protein 37 (RpedOBP37) and cytochrome P450 6a2 (RpedCYP6) throughout developmental stages, tissues, and temperature conditions. These findings are a significant addition to the qRT-PCR analysis studies on R. pedestris, serving as a fundamental groundwork for future investigations on stable reference genes in R. pedestris as well as other organisms.
... The most meaningful transcriptomic measurements rely on relative quantification, that is, the measurement of the expression ratio of target genes to suitable reference genes [23][24][25]. This can, to a certain degree, account for variabilities in sample quality thereby allowing for a higher reproducibility and comparability of experiments [26,27]. Despite the recent cost reduction in high-throughput techniques, such as (single cell) RNA-sequencing and microarrays, these methods are still routinely supported by reverse transcription quantitative real-time PCR (RT-qPCR) for the validation of results and the normalization of data [28]. ...
Prader–Willi syndrome (PWS) is a complex neurodevelopmental disorder caused by the deletion or inactivation of paternally expressed imprinted genes at the chromosomal region 15q11–q13. The PWS-critical region (PWScr) harbors tandemly repeated non-protein coding IPW-A exons hosting the intronic SNORD116 snoRNA gene array that is predominantly expressed in brain. Paternal deletion of PWScr is associated with key PWS symptoms in humans and growth retardation in mice (PWScr model). Dysregulation of the hypothalamic–pituitary axis (HPA) is thought to be causally involved in the PWS phenotype. Here we performed a comprehensive reverse transcription quantitative PCR (RT-qPCR) analysis across nine different brain regions of wild-type (WT) and PWScr mice to identify stably expressed reference genes. Four methods (Delta Ct, BestKeeper, Normfinder and Genorm) were applied to rank 11 selected reference gene candidates according to their expression stability. The resulting panel consists of the top three most stably expressed genes suitable for gene-expression profiling and comparative transcriptome analysis of WT and/or PWScr mouse brain regions. Using these reference genes, we revealed significant differences in the expression patterns of Igfbp7, Nlgn3 and three HPA associated genes: Pcsk1, Pcsk2 and Nhlh2 across investigated brain regions of wild-type and PWScr mice. Our results raise a reasonable doubt on the involvement of the Snord116 in posttranscriptional regulation of Nlgn3 and Nhlh2 genes. We provide a valuable tool for expression analysis of specific genes across different areas of the mouse brain and for comparative investigation of PWScr mouse models to discover and verify different regulatory pathways affecting this complex disorder.
... However, reliable quantification of gene expression using RT-qPCR is dependent on various factors, including the integrity and yield of RNA, efficiency of cDNA synthesis and PCR amplification, primer efficiency, difference in the initial sample amount, and variation in transcriptional activity in the investigated cells and tissues [30][31][32]. Reference genes, whose expression levels remain constant, at least ideally, regardless of experimental treatment, developmental stages, or type of tissues or cells, were therefore used as an internal control to normalize the variations of gene expression data brought by these variables in RT-qPCR analysis [30,[33][34][35][36]. ...
... Selection of proper reference genes that exhibit minimal changes in expression during a specific experiment is crucial for the accuracy of RT-qPCR analysis. However, past studies often selected reference genes based on assumptions rather than evidence [35,37]. Genes involved in basic cellular processes, such as cell structure and primary metabolism, referred to as 'housekeeping genes' , were presumed to have stable expression across tissues, cell types, and experimental conditions. ...
... A high degree of expressional variation among these genes was reported in diverse plant species, including Arabidopsis [38], rice [42], maize [43], tomato [44,45], wheat [46], and poplar [47]. In addition, it has become clear that there are no universal reference genes that would work for all plant species, all tissue types, and different experimental conditions [30,35,48]. Therefore, it is necessary to first identify and validate reference genes with low expressional variation for each species of interest and for the intended research, before carrying out gene expression analysis with RT-qPCR [35,49,50]. ...
Background
Grapevine, as an essential fruit crop with high economic values, has been the focus of molecular studies in diverse areas. Two challenges exist in the grapevine research field: (i) the lack of a rapid, user-friendly and effective RNA isolation protocol for mature dark-skinned berries and, (ii) the lack of validated reference genes that are stable for quantification of gene expression across desired experimental conditions. Successful isolation of RNA with sufficient yield and quality is essential for downstream analyses involving nucleic acids. However, ripe berries of dark-skinned grape cultivars are notoriously challenging in RNA isolation due to high contents of polyphenolics, polysaccharides, RNase and water.
Results
We have optimized an RNA isolation protocol through modulating two factors at the lysis step that could impact results of RNA isolation - 2-ME concentration and berry mass. By finding the optimal combination among the two factors, our refined protocol was highly effective in isolating total RNA with high yield and quality from whole mature berries of an array of dark-skinned wine grape cultivars. Our protocol takes a much shorter time to complete, is highly effective, and eliminates the requirement for hazardous organic solvents. We have also shown that the resulting RNA preps were suitable for multiple downstream analyses, including the detection of viruses and amplification of grapevine genes using reverse transcription-polymerase chain reaction (RT-PCR), gene expression analysis via quantitative reverse transcription PCR (RT-qPCR), and RNA Sequencing (RNA-Seq). By using RNA-Seq data derived from Cabernet Franc, we have identified seven novel reference gene candidates (CYSP, NDUFS8, YLS8, EIF5A2, Gluc, GDT1, and EF-Hand) with stable expression across two tissue types, three developmental stages and status of infection with grapevine leafroll-associated virus 3 (GLRaV-3). We evaluated the stability of these candidate genes together with two conventional reference genes (actin and NAD5) using geNorm, NormFinder and BestKeeper. We found that the novel reference gene candidates outperformed both actin and NAD5. The three most stable reference genes were CYSP, NDUFS8 and YSL8, whereas actin and NAD5 were among the least stable. We further tested if there would be a difference in RT-qPCR quantification results when the most stable (CYSP) and the least stable (actin and NAD5) genes were used for normalization. We concluded that both actin and NAD5 led to erroneous RT-qPCR results in determining the statistical significance and fold-change values of gene expressional change.
Conclusions
We have formulated a rapid, safe and highly effective protocol for isolating RNA from recalcitrant berry tissue of wine grapes. The resulting RNA is of high quality and suitable for RT-qPCR and RNA-Seq. We have identified and validated a set of novel reference genes based on RNA-Seq dataset. We have shown that these new reference genes are superior over actin and NAD5, two of the conventional reference genes commonly used in early studies.
... RT-qPCR is an effective method that provides sensitive and specific quantification of nucleic acids (Chapman and Waldenström 2015). The general steps performed in the RT-qPCR can be listed as RNA isolation, cDNA Synthesis, and RT-qPCR (Nestorov et al. 2013). RT-qPCR was performed by Applied Biosystems (StepOne& StepOnePlus Real-Time PCR Systems) using the TaqMan Primer Probe (Thermo Fisher Scientific) for SP1, SP3 and GAPDH (as housekeeping) genes. ...
Anaemia and chronic obstructive pulmonary disease (COPD) are the common blood and respiratory disorders respectively. The proper lung function is maintained by the SERPINA1 gene predominantly that encodes for alpha 1 antitrypsin protein, which also regulates the iron homeostasis of the human body, whereas imbalance in the iron homeostasis may result in the anaemic condition. The altitudinal variations influence anaemia and COPD. DNA methylation is involved in the early developmental processes, which influences the gene functioning without altering the sequence. The current study has been aimed at analyzing the inter-relationship between anaemia and COPD with DNA methylation of the SERPINA1 gene under altitudinal changes. The methodology involves the DNA isolation, bisulfite conversion and sequencing of the SERPINA1 gene. The results of the current study have shown that SERPINA1 DNA methylation did not significantly involve anaemia and COPD irrespective altitudes, but 3 novel CpG sites cg94377701, cg94389678 and cg94389930 were identified in the SERPINA1 gene of anaemia and COPD patients.
... RT-qPCR is an effective method that provides sensitive and specific quantification of nucleic acids (Chapman and Waldenström 2015). The general steps performed in the RT-qPCR can be listed as RNA isolation, cDNA Synthesis, and RT-qPCR (Nestorov et al. 2013). RT-qPCR was performed by Applied Biosystems (StepOne& StepOnePlus Real-Time PCR Systems) using the TaqMan Primer Probe (Thermo Fisher Scientific) for SP1, SP3 and GAPDH (as housekeeping) genes. ...
The study aimed to determine the expressions of SP1 and SP3 and the clinical-pathological characteristics of patients, and the correlation between the expressions of Specificity Protein 1 (SP1) and Specificity Protein 3 (SP3) in colorectal cancer (CRC). In this study, tumour and adjacent non-tumour tissue samples were obtained from 41 individuals with CRC. SP1 and SP3 expressions were performed using Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR). According to the results of our study, there was no statistically significant difference in SP1 and SP3 expression levels between tumour tissues and non-tumour tissues (p>0.05), as well as no association with clinical-pathological features of patients. In addition, a high positive correlation was found between the expressions of SP1 and SP3 genes in CRC (p=0.01). Consequently, it can be said that there is a correlation between SP1 and SP3 expressions, but SP1 and SP3 expressions are not related to CRC carcinogenesis.
... The method of the first choice for quantitative gene expression analysis is qPCR, which is fast, accurate, economical, and can be easily implemented in a laboratory [15]. However, it is important to normalize qPCR data to compensate for the variability that can appear at all stages, from the quantity and quality of RNA, through reverse transcription to the efficiency of the PCR reaction, and using reference genes [33]. Owing to the high sensitivity of qPCR, normalization with stable reference genes is important for accurate analysis of the biological variation in the data. ...
Delivery of putative compounds of therapeutic value to the brain is limited by brain barriers: the blood–brain barrier located in the endothelium of the brain microvessels (BrMVs) and the blood–cerebrospinal fluid barrier located in the epithelium of the choroid plexus (ChP). Understanding their function and modulation by the circadian clock may enhance the efficacy of brain-targeting therapies. The aim of the present study was to evaluate the stability of 10 reference genes in the BrMV and ChP, isolated from male and female rats at six time points (ZT1, 5, 9, 13, 17, and 21). Gene evaluations were performed by qPCR, analyzed by RefFinder tool, and verified by analyzing the expression of the brain and muscle ARNT-like 1 (Bmal1) using the qPCR and digital PCR methods. We identified as the most stable genes for circadian studies tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz) and apolipoprotein E (Apoe) for BrMV, and beta actin (Actb) and hypoxanthine-guanine phosphoribosyltransferase (Hprt1) for ChP. After verification, ribosomal protein (Rps18) was also included as a sufficient reference gene. Additionally, the observed gender difference in the Bmal1 oscillations in both BrMV and ChP suggests that separate studies for each gender are recommended.
... Housekeeping genes, such as glyceraldehyde-3-phosphate dehydrogenase (Gapdh), β-actin (Actb), and α-tubulin (Tuba1a), have been frequently used as reference genes when the expression of sex hormone receptors in the brain has been analyzed by RT-PCR (Camacho-Arroyo et al., 1998;Guerra-Araiza et al., 2000), or real-time PCR (Scudiero and Verderame, 2017). However, selecting appropriate housekeeping genes is essential with the use of quantitative PCR (qPCR) since their expression could change depending on the treatment or tissue analyzed (Derks et al., 2008;Nestorov et al., 2013). ...
Introduction: In the central nervous system (CNS), tibolone actions are mainly modulated through its interaction
with estrogen, progesterone, and androgen receptors. Several studies have reported the expression of sex hormone
receptors in the CNS using the RT-PCR endpoint technique. Although some studies have validated reference
genes for rat brain tissue in different experimental conditions, no suitable reference genes have been
reported in brain tissue from ovariectomized rats treated with tibolone.
Objective: The aim of this investigation was to evaluate the expression of different housekeeping genes in several
brain regions in ovariectomized rats treated with tibolone to determine the stability of a single housekeeping
gene and a combination of two housekeeping genes under these experimental conditions.
Methods: Adult female Sprague-Dawley rats were ovariectomized. Seven days after the surgery, animals were
administered a single dose of vehicle (water) or tibolone (10 mg/kg/weight). Twenty-four hours later, animals
were sacrificed, and the hypothalamus, hippocampus, prefrontal cortex, and cerebellum were dissected. Total
RNA was extracted from these tissues, and RT-qPCR was performed to amplify Ppia, Hprt1, Rpl32, and Gapdh
housekeeping genes.
Results: Ppia was the most stable gene in the hypothalamus and cerebellum, whereas Hprt1 was the most stable
gene in the prefrontal cortex. For the analysis of the combination of two genes, the most stable combination was
Ppia and Hrpt1 for the prefrontal cortex and Ppia and Rpl32 for the cerebellum.
Conclusion: In ovariectomized rats treated with tibolone, Hprt1 and Ppia genes showed high stability as housekeeping
genes for qPCR analysis.
