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Construction of 15K mouse cDNA microarray. cDNA libraries used for EST generations and the number of ESTs collected from each library are shown (Left). ESTs were clustered by their sequence similarity, and representative cDNA clones were selected from each gene cluster. Numbers and sources of cDNA clones in the 15,264 (15K) unique gene set are shown (Right).
Source publication
cDNA microarray technology has been increasingly used to monitor global gene expression patterns in various tissues and cell types. However, applications to mammalian development have been hampered by the lack of appropriate cDNA collections, particularly for early developmental stages. To overcome this problem, a PCR-based cDNA library constructio...
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Context 1
... of a 15K Mouse Developmental cDNA Microarray. To collect ESTs from early mouse embryos, a PCR-based cDNA library construction method was used to derive ESTs on a large scale from E7.5 extraembryonic tissue (16) and preimplantation em- bryos (17). Additional ESTs were generated for this study from E7.5 extraembryonic cDNA libraries (8,088 cDNAs, GenBank nos. AW537829-AW545916), E7.5 embryonic cDNA libraries (1,589 cDNAs, GenBank nos. AW536144-AW537732), and other libraries (13,241 cDNAs, GenBank nos. AW545922- AW559162) (Fig. 1). All of the ESTs were derived from the 3-ends of cDNAs. All-against-all similarity searches by BLAST program (21) classified the total of 52,374 3-ESTs into 15,264 unique genes, which were rearrayed in a set of 96-well microtiter plates (Fig. 1). This 15K clone set contains cDNA inserts of average 1.5 kb and has been sequenced from both 5-and 3-ends to verify clone identity and further characterize individual genes (G.J.K., D. B. Dudekula, Y. Qian, and M.S.H.K. unpublished work). About 3,400 represent known genes, suggesting that one-third to one-half of well-characterized mammalian genes are included. Because the majority of the cDNAs are derived from early mouse embryos and 78% are either completely novel (55%) or are similar but not identical to known genes (23%), the collection provides a unique source for studies of developmental biology and is complementary to other mousehuman EST ...
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... of a 15K Mouse Developmental cDNA Microarray. To collect ESTs from early mouse embryos, a PCR-based cDNA library construction method was used to derive ESTs on a large scale from E7.5 extraembryonic tissue (16) and preimplantation em- bryos (17). Additional ESTs were generated for this study from E7.5 extraembryonic cDNA libraries (8,088 cDNAs, GenBank nos. AW537829-AW545916), E7.5 embryonic cDNA libraries (1,589 cDNAs, GenBank nos. AW536144-AW537732), and other libraries (13,241 cDNAs, GenBank nos. AW545922- AW559162) (Fig. 1). All of the ESTs were derived from the 3-ends of cDNAs. All-against-all similarity searches by BLAST program (21) classified the total of 52,374 3-ESTs into 15,264 unique genes, which were rearrayed in a set of 96-well microtiter plates (Fig. 1). This 15K clone set contains cDNA inserts of average 1.5 kb and has been sequenced from both 5-and 3-ends to verify clone identity and further characterize individual genes (G.J.K., D. B. Dudekula, Y. Qian, and M.S.H.K. unpublished work). About 3,400 represent known genes, suggesting that one-third to one-half of well-characterized mammalian genes are included. Because the majority of the cDNAs are derived from early mouse embryos and 78% are either completely novel (55%) or are similar but not identical to known genes (23%), the collection provides a unique source for studies of developmental biology and is complementary to other mousehuman EST ...
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... August 1, 2000 vol. 97 no. 16 9129 criteria (24) and allows one to identify 720 genes that are expressed differentially between two samples at the 5% signif- icance level by t test (P value 0.05). These genes are shown as spots in a scatter plot of the average signal intensity of each gene (Fig. 2B). Next, the conventional criteria of 2-fold and 10-fold differ- ences were used to subclassify the significantly different genes into six categories. Sixty-one genes that show 10-fold higher expression in placenta than in embryo are classified as placenta- specific genes; 166 genes with expression levels between 2-fold and 10-fold higher in placenta are classified as predominantly expressed in placenta. Similarly, 57 genes with 10-fold greater expression in embryo are classified as embryo-specific, and 253 whose expression levels are between 2-fold and 10-fold higher in embryo are classified as predominantly expressed in embryo. Expression differences of less than 2-fold have usually been considered at the limit of detection in previous analyses; but, by the statistical criteria applied here, significant differences are reproducibly seen for many differences in the range of 1.2-to 2-fold. The range showing statistically significant differences in less than 2-fold includes 62 genes whose expression is higher in placenta and 121 genes whose expression is higher in embryo. The statistically supported indications are particularly promising for the discovery and analysis of genes that are dosage con- trolled. Frequently important in early development, they include genes that are imprinted or X ...
Citations
... cGAS-STING pathway induces type I and type III IFN production in pDCs via the TBK1-IRF3 axis IFN production in pDCs in response to viral stimuli mostly relies on phosphorylation and nuclear translocation of IRF7 (37). In contrast, cGAS-STING pathway has been reported to be operating via phosphorylation, dimerization, and nuclear translocation of IRF3 facilitated by TBK1 (38). To investigate the role of TBK1 in the cGAS-STING pathway in pDCs, we pretreated purified pDCs with the TBK1 inhibitor BX795 or an inhibitory ODN (iODN) that blocks TLR9 signaling. ...
Plasmacytoid dendritic cells (pDCs) are potent producers of type I and type III IFNs and play a major role in antiviral immunity and autoimmune disorders. The innate sensing of nucleic acids remains the major initiating factor for IFN production by pDCs. TLR-mediated sensing of nucleic acids via endosomal pathways has been studied and documented in detail, whereas the sensing of DNA in cytosolic compartment in human pDCs remains relatively unexplored. We now demonstrate the existence and functionality of the components of cytosolic DNA-sensing pathway comprising cyclic GMP-AMP (cGAMP) synthase (cGAS) and stimulator of IFN gene (STING) in human pDCs. cGAS was initially located in the cytosolic compartment of pDCs and time-dependently colocalized with non-CpG double-stranded immunostimulatory DNA (ISD). Following the colocalization of ISD with cGAS, the downstream pathway was triggered as STING disassociated from its location at the endoplasmic reticulum. Upon direct stimulation of pDCs by STING agonist 2'3' cGAMP or dsDNA, pDC-s produced type I, and type III IFN. Moreover, we documented that cGAS-STING-mediated IFN production is mediated by nuclear translocation of IRF3 whereas TLR9-mediated activation occurs through IRF7. Our data also indicate that pDC prestimulation of cGAS-STING dampened the TLR9-mediated IFN production. Furthermore, triggering of cGAS-STING induced expression of SOCS1 and SOCS3 in pDCs, indicating a possible autoinhibitory loop that impedes IFN production by pDCs. Thus, our study indicates that the cGAS-STING pathway exists in parallel to the TLR9-mediated DNA recognition in human pDCs with cross-talk between these two pathways.
... The first high-throughput platforms dedicated to the survey of mRNA were cDNA microarrays developed and used successfully to estimate the transcriptional contents of early embryos (Ko et al. 2000, Tanaka et al. 2000, Carter et al. 2003, Sirard et al. 2005, Misirlioglu et al. 2006, Somers et al. 2006). The study of early mammalian development always has been and remains technically challenging, since embryos are a scant source of biological material. ...
... The first high-throughput platforms dedicated to the survey of mRNA were cDNA microarrays developed and used successfully to estimate the transcriptional contents of early embryos (Ko et al. 2000, Tanaka et al. 2000, Carter et al. 2003, Sirard et al. 2005, Misirlioglu et al. 2006, Somers et al. 2006). The study of early mammalian development always has been and remains technically challenging, since embryos are a scant source of biological material. ...
... The much higher expression level of Rain in macrophage compared with liver (the value of log 2 folded change: 4.2 vs. 3.3) suggested that liver vascular tubulogenesis and fibrosis may be induced by macrophage. Another candidate, lncRNA 1700020I14Rik that also named as Cyrano was seldom investigated, and the effect of this gene on embryonic development was only identified in zebrafish [39]. corresponding controls were shown in (A and B) respectively (p < 0.01), lncRNAs both differentially expressed in macrophages and liver were shown in (C), the log 2 folded change value was used to estimate the lncRNA expression levels before and after silica particle exposure. ...
Liver disorders such as hepatitis, cirrhosis and hepatocellular carcinoma are a series of the most life threatening diseases along with extensive inflammatory cellular infiltrations. Macrophage has been proved to be key regulators and initiators of inflammation, and long non-coding RNAs (lncRNAs) are recommended to play critical roles in the occurrence and development of a variety of diseases. To uncover the role of macrophage in liver disorders via lncRNA sequencing method, we first applied a lncRNA classification pipeline to identify 1247 lncRNAs represented on the Affymetrix Mouse Genome 430/430A 2.0 array. We then analyzed the lncRNA expression patterns in a set of previously published gene expression profiles of silica particle exposed macrophages and liver respectively, and identified and validated sets of differentially expressed lncRNAs shared by macrophages and liver. The functional enrichment analysis of these lncRNAs was processed on the basis of their expression signatures, three aspects including cis, trans and co-acting proteins were proposed. This is the first time to correlate macrophage with liver disorders via co-expressed lncRNAs. Our findings indicated that roles of macrophage in liver disorders were double-edged, the differentially expressed lncRNAs and their corresponding regulatory genes or proteins may serve as potential diagnostic biomarkers and therapeutic targets.
... Knockdown of PLAC8 with siRNAs significantly reduced the proliferation and Electronic supplementary material The online version of this article (doi:10.1007/s11255-017-1578-y) contains supplementary material, which is available to authorized users. Placenta specific 8 (PLAC8), previously called onzin, is a small protein that has been shown to be highly expressed in giant trophoblasts and the spongiotrophoblast layer of the mouse placenta [7,8]. PLAC8 was first identified in human dendritic cells [9]; then, the expression of PLAC8 was subsequently detected in myeloid and lymphoid cells, as well as epithelial cells in the lung and intestine [10]. ...
Background:
Placenta specific 8 (PLAC8) plays an important role in many different cellular processes and human diseases, including multiple types of cancer. However, the functional role of PLAC8 in clear cell renal cell carcinoma (ccRCC) has never been elucidated.
Methods:
PLAC8 mRNA expression was investigated in 31 pairs of fresh ccRCC tumor tissues and matched adjacent non-tumor tissues by RT-qPCR and confirmed by analyzing the TCGA KRCC dataset which contains RNA-seq data of 534 ccRCC and 72 solid normal tissues. Protein level of PLAC8 expression was also investigated using immunohistochemistry in 129 ccRCC samples. Correlations with clinicopathological factors and overall survival were analyzed. To examine its effect on the biological activity, PLAC8 siRNAs were transfected into ccRCC cells. Cell proliferation was assessed by CCK8 cell viability assays, clone formation assays, and EdU incorporation assays. Cell invasion was examined using transwell assays. RNA sequencing was then performed to further elucidate the mechanisms by which PLAC8 regulates the cancer.
Results:
PLAC8 expression was positively correlated with tumor size, metastasis, and clinical stage of ccRCC. Additionally, high PLAC8 expression was closely associated with a shorter overall survival time. Knockdown of PLAC8 with siRNAs significantly reduced the proliferation and invasion of RCC cells and increased the sensitivity of RCC cells to cisplatin. RNA-seq analysis revealed that knockdown of PLAC8 down-regulated the expression of a panel of inflammatory mediators, which suggested that PLAC8 is associated with the ccRCC inflammatory microenvironment. Patients with high expression of PLAC8 had a significantly higher number of infiltrative lymphocytes than patients with low expression of PLAC8.
Conclusion:
Our findings suggest that PLAC8 may be a potential prognostic indicator and therapeutic target for ccRCC.
... Arabidopsis thaliana mRNAs (Stratagene, La Jolla, CA) were introduced as internal probe standards into reverse transcription reactions to normalize data between different arrays. Cy3-or Cy5-fluorescently labeled cDNAs were hybridized onto the National Institute for Aging 15K mouse cDNA prefabricated arrays (Tanaka et al., 2000 ), supplied by the Massachusetts Institute of Technology (MIT)-BioMicro Center, using the procedure provided by the MIT-BioMicro Center. Hybridized microarrays were scanned with the arrayWoRx e ™ Biochip Reader (Applied Precision LLC, Northwest Issaquah, WA). ...
... S1; SupplementaryTable S1). Prefabricated cDNA micro-arrays constructed with the National Institute for Aging mouse 15K mouse clone set were used for the analysis (Tanaka et al., 2000; Kargul et al., 2001). As detailed in Materials and methods, for each of the 4 experimental comparisons, we isolated two independent samples of RNA; and each of the RNA samples was labeled independently with Cy5 and Cy3 fluorescent dyes. ...
There is a long-standing unmet clinical need for biomarkers with high specificity for distributed stem cells (DSCs) in tissues, or for use in diagnostic and therapeutic cell preparations (e.g., bone marrow). Although DSCs are essential for tissue maintenance and repair, accurate determination of their numbers for medical applications has been problematic. Previous searches for biomarkers expressed specifically in DSCs were hampered by difficulty obtaining pure DSCs and by the challenges in mining complex molecular expression data. To identify such useful and specific DSC biomarkers, we combined a novel sparse feature selection method with combinatorial molecular expression data focused on asymmetric self-renewal, a conspicuous property of DSCs. The analysis identified reduced expression of the histone H2A variant H2A.Z as a superior molecular discriminator for DSC asymmetric self-renewal. Subsequent molecular expression studies showed H2A.Z to be a novel "pattern-specific biomarker" for asymmetrically self-renewing cells, with sufficient specificity to count asymmetrically self-renewing DSCs in vitro and potentially in situ.
Copyright © 2015. Published by Elsevier B.V.
... Research on discovering biological, pathological mechanisms through genetic regulation has been increasing since the completion of the genetic map of several species, including that of human beings (453) structure of genes, makes it possible to determine the general expression of an entire gene within a cell. It is a major tool in function genomics research 5,6) . ...
Objectives: cDNA microarray is an effective method to snapshot gene expression. Functional clustering of gene expressions can identify herbal medicine mechanisms. Much microarray data is available for various herbal medicines. This study compares regulated genes with herbal medicines to evaluate the nature of the drugs. Methods: Published microarray data were collected. Total RNAs were prepared from dissociated hippocampal dissociate cultures which were given hypoxic shock in the presence of each herbal medicine. Up- or downregulated genes higher than Global M value 0.5 were selected, clustered in functional groups, and compared with various herbal treatments. Results: 1. Akt2 was upregulated by Acorus gramineus SOLAND, Arisaema amurense var. serratum N AKAI and Coptis chinensis FRANCH, and they belong to Araceae herb. 2. Nf-κb1, Cd5, Gnγ7 and Sgne1 were upregulated by Arisaema amurense var. serratum NAKAI, Coptis chinensis F RANCH and Rheum coreanum N AKAI . 3. Woohwangcheongsim-won, Sohaphyang-won and Scutellaria baicalensis GEORGI downregulated Scp2 and upregulated Tsc2. Woohwangcheongsim-won and Sohaphyang-won upregulated Hba1 and downregulated Myf6. 4. Sohaphyang-won and Scutellaria baicalensis GEORGI downregulated Slc12a1. 5. Woohwangcheongsim-won and Arisaema amurense var. serratum NAKAI upregulated Rarα, Woohwangcheongsim-won and Coptis chinensis F RANCH downregulated Rab5a and Pdgfrα, and Woohwangcheongsim-won and Rheum coreanum NAKAI upregulated Plcγ1 and downregulated Pla2g1b and Slc10a1. Conclusions: By clustering microarray, genes are commonly identified to be either up- or downregulated. These results will provide new information to understand the efficacy of herbal medicines and to classify them at the molecular level.
... After labelling, repetitive sequences within the cDNA samples were blocked with 10 μg mouse Cot-1 DNA (Invitrogen) to prevent non-specific sequences binding to the cDNA probes. Cy5labelled test and Cy3-labelled reference were co-hybridised onto custom cDNA mouse microarrays containing 13,825 features (NIA 15 K Mouse cDNA clone set) [104]. Three total RNA samples from 3 mice were hybridised with UMRR. ...
Background
Gene expression changes induced by carcinogens may identify differences in molecular function between target and non-target organs. Target organs for benzo[a]pyrene (BaP) carcinogenicity in mice (lung, spleen and forestomach) and three non-target organs (liver, colon and glandular stomach) were investigated for DNA adducts by 32P-postlabelling, for gene expression changes by cDNA microarray and for miRNA expression changes by miRNA microarray after exposure of animals to BaP.
Results
BaP-DNA adduct formation occurred in all six organs at levels that did not distinguish between target and non-target. cDNA microarray analysis showed a variety of genes modulated significantly by BaP in the six organs and the overall gene expression patterns were tissue specific. Gene ontology analysis also revealed that BaP-induced bioactivities were tissue specific; eight genes (Tubb5, Fos, Cdh1, Cyp1a1, Apc, Myc, Ctnnb1 and Cav) showed significant expression difference between three target and three non-target organs. Additionally, several gene expression changes, such as in Trp53 activation and Stat3 activity suggested some similarities in molecular mechanisms in two target organs (lung and spleen), which were not found in the other four organs. Changes in miRNA expression were generally tissue specific, involving, in total, 21/54 miRNAs significantly up- or down-regulated.
Conclusions
Altogether, these findings showed that DNA adduct levels and early gene expression changes did not fully distinguish target from non-target organs. However, mechanisms related to early changes in p53, Stat3 and Wnt/β-catenin pathways may play roles in defining BaP organotropism.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-880) contains supplementary material, which is available to authorized users.
... The scarcity of the mRNA amounts obtained from preimplantation embryos has hampered the molecular analysis of preimplantation embryos [13][14][15]. Recent progress in RNA amplification methods and microarray platforms, including genes unique to preimplantation embryos, allow us to apply global gene expression profiling to the study of preimplantation embryos [16][17][18][19]. Initially, the majority of studies focused on gene expression analysis of preimplantation embryos, which were based on conventional reverse-transcriptase polymerase chain reaction (RT-PCR) [20,21]. ...
Background
Real-time quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) is the most sensitive, and valuable technique for rare mRNA detection. However, the expression profiles of reference genes under different experimental conditions, such as different mouse strains, developmental stage, and culture conditions have been poorly studied.
Results
mRNA stability of the actb, gapdh, sdha, ablim, ywhaz, sptbn, h2afz, tgfb1, 18 s and wrnip genes was analyzed. Using the NormFinder program, the most stable genes are as follows: h2afz for the B6D2F-1 and C57BL/6 strains; sptbn for ICR; h2afz for KOSOM and CZB cultures of B6D2F-1 and C57BL/6 strain-derived embryos; wrnip for M16 culture of B6D2F-1 and C57BL/6 strain-derived embryos; ywhaz, tgfb1, 18 s, 18 s, ywhaz, and h2afz for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst embryonic stages cultured in KSOM medium, respectively; h2afz, wrnip, wrnip, h2afz, ywhaz, and ablim for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in CZB medium, respectively; 18 s, h2afz, h2afz, actb, h2afz, and wrnip for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in M16 medium, respectively.
Conclusions
These results demonstrated that candidate reference genes for normalization of target gene expression using RT-qPCR should be selected according to mouse strains, developmental stage, and culture conditions.
Electronic supplementary material
The online version of this article (doi:10.1186/1756-0500-7-675) contains supplementary material, which is available to authorized users.
... Selection of the unique gene set was done by all-against-all similarity searches with BLAST algorithm with a cutoff score of 800 to group expressed sequence tags (ESTs) into unique genes, as previously described (Tanaka et al., 2000). The array consisted of 20,736 mouse cDNA clones representing approximately a total of 15,000 unique genes or UniGene clusters. ...
... The length of cDNA inserts was in the range of 300–1200 bp, with a mean value of ∼750 bp. cDNA library constructions were based on oligo-dT–primed reverse transcription of total RNA (Tanaka et al., 2000). After dehydration, PCR products were resuspended in the spotting buffer (75% formamide and 25% water) and spotted in duplicate onto Ultra-GAPSTM amino-silane–coated slides (Corning BV, Schiphol-Rik, The Netherlands) using a MicroGrid II Arrayer (BioRobotics, Cambridge, UK). ...
Fenofibrate is a PPARα ligand that has been widely used as a lipid lowering agent in the treatment of hypertriglyceridemia. ABCD2 (D2) is a peroxisomal long-chain acyl-CoA transporter that is highly induced by fenofibrate in the livers of mice. To determine if D2 is a modifier of fibrate responses, wild-type and D2 deficient mice were treated with fenofibrate for 14 days. The absence of D2 altered expression of gene clusters associated with lipid metabolism, including PPARα signaling. Using 3T3-L1 adipocytes, which express high levels of D2, we confirmed that knock-down of D2 modified genomic responses to fibrate treatment. We next evaluated the impact of D2 on effects of fibrates in a mouse model of diet-induced obesity. Fenofibrate treatment opposed the development of obesity, hypertriglyceridemia, and insulin resistance. However, these effects were unaffected by D2 genotype. We concluded that D2 can modulate genomic responses to fibrates, but that these effects are not sufficiently robust to alter the effects of fibrates on diet-induced obesity phenotypes.
... Preimplantation development is important to reproductive biology applications including livestock breeding and treatments that repair defective tissues (Hamatani et al., 2004; Wang and Dey, 2006; Chakrabarty et al., 2007), and developmental biology may be studied with genomics (Ko, 2001), Canestro et al. (2007).More specifically we can learn from changes in gene expression during preimplantation development in the mouse (Tanaka et al., 2000; Zeng and Schultz, 2003; Wobus and Boheler 2005). This research note describes statistical analysis and results of global gene expression profiles at preimplantation development stages. ...
