MiRNA profiling in hPASMC isolated from normal and PAH patients
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Guofei Zhou
PROVIDER: E-GEOD-55427 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Normal fibroblasts and SSc fibroblasts between the third and six subpassages were used for experiments. Total RNA was extracted from culture cells with ISOGEN (Nippon Gene, Tokyo, Japan). MicroRNA isolation from total RNA was performed using RT2 qPCR-Grade miRNA Isolation Kit (SA Bioscience). For RT2 Profiler PCR Array (SABioscience), microRNAs were reverse-transcribed into first strand cDNA using RT2 miRNA First Strand Kit (SABiosciences). A mixture of equal amounts of cDNAs from 5 normal fibroblasts or 5 SSc fibroblasts was prepared. The cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix and the mixture was added into a 96-well RT2 miRNA PCR Array (SABiosciences) that included primer pairs for 88 human microRNAs. Human dermal fibroblasts were obtained by skin biopsy from the affected areas (dorsal forearm) of 5 patients with diffuse cutaneous SSc and <2 years of skin thickening. Control fibroblasts were obtained by skin biopsies from 5 healthy donors. Control donors were each matched with a SSc patient for age, sex, and biopsy site.
2012-01-01 | E-GEOD-34142 | biostudies-arrayexpress
Project description:Normal fibroblasts and SSc fibroblasts between the third and six subpassages were used for experiments. Normal and scleroderma fibroblasts were serum-starved for 24 hours and incubated in the presence or absence of TGF-β1 (2ng/ml) for 6 hours. Total RNA was extracted from culture cells with ISOGEN (Nippon Gene, Tokyo, Japan). MicroRNA isolation from total RNA was performed using RT2 qPCR-Grade miRNA Isolation Kit (SA Bioscience). For RT2 Profiler PCR Array (SABioscience), microRNAs were reverse-transcribed into first strand cDNA using RT2 miRNA First Strand Kit (SABiosciences). A mixture of equal amounts of cDNAs from 5 normal fibroblasts or 5 SSc fibroblasts was prepared. The cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix and the mixture was added into a 96-well RT2 miRNA PCR Array (SABiosciences) that included primer pairs for 88 human microRNAs. Human dermal fibroblasts were obtained by skin biopsy from the affected areas (dorsal forearm) of 5 patients with diffuse cutaneous SSc and <2 years of skin thickening. Control fibroblasts were obtained by skin biopsies from 5 healthy donors. Control donors were each matched with a SSc patient for age, sex, and biopsy site.
2012-04-01 | E-GEOD-34827 | biostudies-arrayexpress
Project description:Our previous findings suggest that the nucleus of the solitary tract (NTS), a pivotal region for regulating the set-point of arterial pressure, exhibits abnormal inflammation in pre-hypertensive and spontaneously hypertensive rats (SHRs) together with elevated anti-apoptotic and low apoptotic factor levels compared with that of normotensive Wistar–Kyoto (WKY) rats. Whether this chronic condition affects neuronal growth and plasticity in the NTS remains unknown. To unveil the characteristics of the neurodevelopmental environment in the NTS of hypertensive rats, we investigated the gene expression profile of neurotrophins and their receptors in SHRs compared to that of normotensive rat WKY. The NTS was dissected from the brain of 6 SHRs and 6 WKY rats and the total RNA was extracted. In both groups of rats (SHRs & WKY rats, n = 6 each), a total of 2 ug mRNA extracts from each NTS were pooled together, treated with RNase-free DNAse I (Invitrogen Life technologies) to remove any genomic contamination, and further purified using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. Reverse transcription was subsequently performed on 1 ug total RNA using SuperArray’s RT2 First Strand Kit (SABiosciences); the resulting cDNA was submitted for real-time quantitative PCR reactions on RT2 ProfilerTM PCR array plates using Superarray RT2 SYBR Green qPCR Master Mix (SAbiosciences) and iCycler iQ thermal cycler (Bio-rad), following the manufacturer’s instructions. The experiment was performed in duplicate in each group.
2015-04-15 | E-GEOD-67874 | biostudies-arrayexpress
Project description:Real-time quantitative PCR analysis of human epithelial cells The activity of Malva sylvestris extract and fractions infected by A. actinomycetemcomitans were investigated using an adapted dual chamber model, oral human epithelial cells were used in the co-culture model. One microgram of RNA was converted in cDNA using RT2 First Strand Kit. 84 genes were analyzed using inflammatory response & Autoimmunity Array RT2 profiler (Qiagen Sabiosciences, Valencia, CA, USA) with buffers supplied by the manufacturer qPCR gene expression profiling. Oral human epithelial cells were infected by A. Actinomycetemcomintans and treated with Malva sylvestris extract and fractions prior to gene expression analysis.
2015-08-27 | E-GEOD-72443 | biostudies-arrayexpress
Project description:Human DNA Damage Signaling Pathway RT2 Profiler Array Kit (Qiagen, Valencia, CA) was used to profile the expression of key genes involved in the DDR network as per the manufacturerâs instructions. U937 cells were incubated with PXD-101 +/- MLN4924 for 16 h, after which the RT2 ProfilerTM DNA damage Signaling Pathway PCR Array was performed to profile the expression of 84 human genes involved in the DDR network.
2016-02-05 | E-GEOD-77560 | biostudies-arrayexpress
Project description:RNA was isolated with the RNeasy kit (Qiagen) from electronically sorted total CD4SP thymocytes. RNA was reverse transcribed into cDNA by oligo(dT) priming with the QuantiTect Reverse transcription kit (Qiagen). The expression of 84 key genes involved in apoptosis, or programmed cell death was performed with mouse apoptosis RT2ProfilerTM PCR array system (PAMM-012 E; SABiosciences). PCR Array data were analyzed by the Ct data analysis method. qPCR gene expression profiling. CD4SP thymocytes from WT and Ik7Tg mice were used.
2013-07-27 | E-GEOD-49264 | biostudies-arrayexpress
Project description:RNA was isolated with the RNeasy kit (Qiagen) from electronically sorted immature CD4SP thymocytes. RNA was reverse transcribed into cDNA by oligo(dT) priming with the QuantiTect Reverse transcription kit (Qiagen). The expression of 84 key genes involved in apoptosis, or programmed cell death was performed with mouse apoptosis RT2ProfilerTM PCR array system (PAMM-012 E; SABiosciences). PCR Array data were analyzed by the Ct data analysis method. qPCR gene expression profiling. CD4SP thymocytes from WT and Ik7Tg mice were used.
2013-07-27 | E-GEOD-49270 | biostudies-arrayexpress
Project description:RNA was isolated with the RNeasy kit (Qiagen) from electronically sorted mature CD4SP thymocytes. RNA was reverse transcribed into cDNA by oligo(dT) priming with the QuantiTect Reverse transcription kit (Qiagen). The expression of 84 key genes involved in apoptosis, or programmed cell death was performed with mouse apoptosis RT2ProfilerTM PCR array system (PAMM-012 E; SABiosciences). PCR Array data were analyzed by the Ct data analysis method. qPCR gene expression profiling. CD4SP thymocytes from WT and Ik7Tg mice were used.
2013-07-27 | E-GEOD-49271 | biostudies-arrayexpress
Project description:We previously demonstrated that the osteopontin-c (OPNc) splice variant activates several aspects of the progression of prostate cancers. The goal of the present study was to develop cell line model to determine the impact of OPNc overexpression on main cancer signaling pathways and thus obtain insights into the mechanisms of OPNc pro-tumorigenic roles. Methods: PC-3 cells were stably transfected to overexpress OPNc. Transcriptomic profiling was performed on these cells and compared to controls, to identify OPNc overexpression-dependent changes in gene expression levels and pathways by qRT-PCR analyses; Results: Of 84 genes tested by using a multiplex real-time PCR Cancer Pathway Array approach, among then 16 were differentially expressed between PC-3 OPNc-overexpressing cells in relation to control clones. Differentially expressed genes are included in all main hallmarks of cancer, and several interacting proteins have been identified using an interactome network analysis. Conclusions: Overall, the present study elucidated transcriptional changes of prostate cancer cells in response to OPNc overexpression, which provides an assessment for predicting the molecular mechanisms by which this splice variant promotes tumor progression features. Cell Culture, OPN Plasmids and Transfection; As a model to examine the signaling pathways modulated by OPNc overexpression in prostate carcinoma, we used PC-3 cell line, which were provided by ATCC. The open reading frame of OPNc was cloned into the pCR3.1 mammalian expression vector as previously described [Tilli et al., 2012]. Transfections were performed using LipofectamineTM 2000 (Invitrogen, CA). Our previous data demonstrated that PC-3 stably transfected cells contain higher levels of protein and the mRNA OPNc splicing isoform in relation to their endogenous levels in empty vector-transfected cells. PC-3 transfected with empty vector (EV) were used as a negative control in these assays. Human Cancer Pathway Finder PCR Array The Human Cancer Pathway Finder SuperArray (PAHS-033A; Qiagen) was used to determine changes in the specific genes encoding proteins related to the main hallmarks of cancer in response to OPNc overexpression. The assay design criteria ensure that each qPCR reaction will generate single, gene-specific amplicons and prevent the co-amplification of non-specific products. The qPCR Assays used in PCR Arrays are optimized to work under standard conditions enabling a large number of genes to be assayed simultaneously. Their specificity is guaranteed by SABiosciences when RT2 SYBR Green qPCR Master Mixes are used as part of the complete PCR Array System protocol. Simultaneous gene expression analyses require similar qPCR efficiencies for accurate comparison among genes. RT2 qPCR Primer Assays are designed with an amplicon size ranging from 100 to 250 bp and with PCR efficiencies uniformly greater than 90%. Overall, more than 10 thermodynamic criteria are included in the design of each RT2 qPCR Primer Assay to ensure the most reliable and accurate results for pathway-based gene expression analysis in the PCR Array System. We analyzed mRNA levels of 84 genes related to cell cycle control, apoptosis and cell senescence, signal transduction molecules and transcription factors, adhesion, angiogenesis, invasion and metastasis; and also 5 housekeeping genes and genomic DNA contamination controls. The PCR plates were run using the CFX96 Real-Time System cycler (BioRad, Hercules, CA), following a superarray two-step cycling PCR protocol, in which each plate ran one cycle for 10 minutes at 95M-BM-0C, as well as 40 cycles of 95M-BM-0C for 15 seconds and 60M-BM-0C for 1 minute. After the superarray protocol was run for each plate, RT-PCR data analysis was performed using the website:http://www.SABiosciences.com/pcrarraydataanalysis.php, in order to compare gene expression of OPNc-overexpressing cells and empty vector transfected cells. Total RNA quality control, cDNA synthesis and the quantitative real-time RT-PCR (qRT-PCR) array were performed as recommended by the manufacturer (Qiagen). Data analysis of gene expression was performed using Excel-based PCR Array Data Analysis Software provided by manufacturer (Qiagen). Fold-changes in gene expression were calculated using the M-NM-^TM-NM-^TCT method, and five stably expressed housekeeping genes (M-NM-22 microglobulin, hypoxanthine phosphoribosyltransferase 1, ribosomal protein L13a, GAPDH and M-NM-2-actin) were used to normalize the level of expression. The statistical analysis was performed to compare the gene expression values for the OPNc-overexpressing cells and those transfected with empty vector. P<0.05 was considered statistically significant. Only genes showing a 1.5-fold or greater change were considered for further analysis.
2014-05-23 | E-GEOD-57889 | biostudies-arrayexpress
Project description:We previously demonstrated that the osteopontin-c (OPNc) splice variant activates several aspects of the progression of ovarian cancers. The goal of the present study was to develop cell line model to determine the impact of OPNc overexpression on main cancer signaling pathways and thus obtain insights into the mechanisms of OPNc pro-tumorigenic roles. Methods: OvCar-3 cells were stably transfected to overexpress OPNc. Transcriptomic profiling was performed on these cells and compared to controls, to identify OPNc overexpression-dependent changes in gene expression levels and pathways by qRT-PCR analyses; Results: Of 84 genes tested by using a multiplex real-time PCR Cancer Pathway Array approach, 34, were differentially expressed between OvCar-3 OPNc-overexpressing cells in relation to control clones. Differentially expressed genes are included in all main hallmarks of cancer, and several interacting proteins have been identified using an interactome network analysis. Conclusions: Overall, the present study elucidated transcriptional changes of ovarian cancer cells in response to OPNc overexpression, which provides an assessment for predicting the molecular mechanisms by which this splice variant promotes tumor progression features. Cell Culture, OPN Plasmids and Transfection; As a model to examine the signaling pathways modulated by OPNc overexpression in ovarian carcinoma, we used OvCar-3 cell line, which were provided by ATCC. The open reading frame of OPNc was cloned into the pCR3.1 mammalian expression vector as previously described [Tilli et al., 2012]. Transfections were performed using LipofectamineTM 2000 (Invitrogen, CA). Our previous data demonstrated that OvCar-3 stably transfected cells contain higher levels of protein and the mRNA OPNc splicing isoform in relation to their endogenous levels in empty vector-transfected cells. OvCar-3 transfected with empty vector (EV) were used as a negative control in these assays. Human Cancer Pathway Finder PCR Array The Human Cancer Pathway Finder SuperArray (PAHS-033A; Qiagen) was used to determine changes in the specific genes encoding proteins related to the main hallmarks of cancer in response to OPNc overexpression. The assay design criteria ensure that each qPCR reaction will generate single, gene-specific amplicons and prevent the co-amplification of non-specific products. The qPCR Assays used in PCR Arrays are optimized to work under standard conditions enabling a large number of genes to be assayed simultaneously. Their specificity is guaranteed by SABiosciences when RT2 SYBR Green qPCR Master Mixes are used as part of the complete PCR Array System protocol. Simultaneous gene expression analyses require similar qPCR efficiencies for accurate comparison among genes. RT2 qPCR Primer Assays are designed with an amplicon size ranging from 100 to 250 bp and with PCR efficiencies uniformly greater than 90%. Overall, more than 10 thermodynamic criteria are included in the design of each RT2 qPCR Primer Assay to ensure the most reliable and accurate results for pathway-based gene expression analysis in the PCR Array System. We analyzed mRNA levels of 84 genes related to cell cycle control, apoptosis and cell senescence, signal transduction molecules and transcription factors, adhesion, angiogenesis, invasion and metastasis; and also 5 housekeeping genes and genomic DNA contamination controls. The PCR plates were run using the CFX96 Real-Time System cycler (BioRad, Hercules, CA), following a superarray two-step cycling PCR protocol, in which each plate ran one cycle for 10 minutes at 95M-BM-0C, as well as 40 cycles of 95M-BM-0C for 15 seconds and 60M-BM-0C for 1 minute. After the superarray protocol was run for each plate, RT-PCR data analysis was performed using the website:http://www.SABiosciences.com/pcrarraydataanalysis.php, in order to compare gene expression of OPNc-overexpressing cells and empty vector transfected cells. Total RNA quality control, cDNA synthesis and the quantitative real-time RT-PCR (qRT-PCR) array were performed as recommended by the manufacturer (Qiagen). Data analysis of gene expression was performed using Excel-based PCR Array Data Analysis Software provided by manufacturer (Qiagen). Fold-changes in gene expression were calculated using the M-NM-^TM-NM-^TCT method, and five stably expressed housekeeping genes (M-NM-22 microglobulin, hypoxanthine phosphoribosyltransferase 1, ribosomal protein L13a, GAPDH and M-NM-2-actin) were used to normalize the level of expression. The statistical analysis was performed to compare the gene expression values for the OPNc-overexpressing cells and those transfected with empty vector. P<0.05 was considered statistically significant. Only genes showing a 1.5-fold or greater change were considered for further analysis.
2014-05-23 | E-GEOD-57888 | biostudies-arrayexpress