Reproducibility of Quantitative RT-PCR Array in miRNA Expression Profiling and Comparison with Microarray Analysis
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE16000 | GEO | 2009/05/13
SECONDARY ACCESSION(S): PRJNA115415
REPOSITORIES: GEO
Similar Datasets
Project description:High reproducibility with TaqMan microRNA array (qPCR-array) was demonstrated by comparing replicate results from the same RNA sample. Pre-amplification of the miRNA cDNA improved sensitivity of the qPCR-array and increased the number of detectable miRNAs. Furthermore, the relative expression levels of miRNAs were maintained after pre-amplification. When the performance of qPCR-array and microarrays were compared using different aliquots of the same RNA, a low correlation between the two methods (r = -0.443) indicated considerable variability between the two assay platforms. Higher variation between replicates was observed in miRNAs with low expression in both assays. Finally, a higher false positive rate of differential miRNA expression was observed using the microarray compared to the qPCR-array. Replicate preparations (n =4) using different aliquots of a single C2C12 RNA sample (500 ng) were used to determine the reproducibility of the reverse transcription process as well as the results of the same reverse transcription products performed on different days. TaqMan microRNA Array A was used for this purpose. To assess the reliability of pre-amplification, miRNA expression profiles obtained with and without pre-amplification using MiRNA TaqMan Array B were performed. Independent miRNA expression profiling studies using uParaflo microfluidic biochips were performed by an independent company to determine the relationship between results obtained with qPCR-array and microarrays. Aliquots of the same C2C12 RNA were used in both the qPCR-array (500 ng) and microarrays (8 µg).
2010-05-19 | E-GEOD-16000 | biostudies-arrayexpress
Project description:Mutations in SOD1 (Superoxide Dismutase 1) gene are associated with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. By employing ascorbate peroxidase (APEX)-based proximity labeling, coupled with LC-MS/MS analysis, we uncovered 37 and 28 proteins exhibiting higher abundance in the proximity proteomes of SOD1G85R and SOD1G93A, respectively, than that of the wild-type SOD1. Immunoprecipitation followed by western blot analysis confirmed the preferential binding of one of these proteins, exportin 5 (XPO5), toward the two mutants of SOD1 over its wild-type counterpart. In line with the established function of XPO5 in pre-miRNA transport, we observed diminished nucleocytoplasmic transport of pre-miRNAs in cells with ectopic expression of the two SOD1 mutants over those expressing the wild-type counterpart. On the other hand, RT-qPCR results revealed significant elevations in mature miRNA in cells expressing the two SOD1 mutants, which are attributed to diminished inhibitory effect of XPO5 on Dicer-mediated cleavage of pre-miRNA to mature miRNA. Together, our chemoproteomic approach led to the revelation of a novel mechanism through which ALS-associated mutants of SOD1 perturb miRNA biogenesis, i.e., through aberrant binding toward XPO5.
2024-01-26 | PXD035452 | Pride
Project description:Five aliquots of the same RNA sample were labeled and hybridized in parallel to the L. vannamei microarray, in order to assess the technical reproducibility of the tool Keywords: technical validation
2006-06-01 | GSE4954 | GEO
Project description:Hairpin-containing pre-miRNAs are precursors of microRNAs (miRNAs) that play important roles in cellular processes and various human diseases. Pre-miRNAs are produced from longer primary transcripts (pri-miRNAs). The falsity in cellular expression and sequences of pre-miRNAs might cause cellular defects or human diseases. However, the current pre-miRNA quantification methods by qPCR cannot discriminate between pre-miRNAs and their parental pri-miRNAs. In addition, the ligation of the sequencing adapter to 5’-end of pre-miRNAs is inefficient, therefore, pre-miRNA sequencing is highly impractical. Here, we developed a method, called the intramolecular ligation method (iLIME) for pre-miRNA quantification and sequencing. This method utilized T4 RNA ligase 1 to convert hairpin-pre-miRNAs into circularized RNAs that do not naturally exist in cells. The resulting circuliarized RNAs allow us to design unique primers to quantify pre-miRNAs by qPCR specicially, and thus this qPCR can distinguish pre-miRNA from pri-miRNAs. In addition, the iLIME also allows us to sequence pre-miRNAs using next-generation sequencing. The iLIME method offers a simple and effective way to quantify and sequence pre-miRNAs. This will be useful in investigating pre-miRNAs for addressing research questions for medical applications. The iLIME can be potentially extended to other hairpin-containing RNAs, such as tRNAs and snRNAs.
2021-10-11 | GSE185524 | GEO
Project description:Five aliquots of the same RNA sample were labeled and hybridized in parallel to the L. vannamei microarray, in order to assess the technical reproducibility of the tool Keywords: technical validation A sample of gill total RNA was divided in 5 identical aliquots, and each aliquot was labeled and hybridized to an array. The co-relation among the global expression profiles obtained was assessed.
2010-07-01 | E-GEOD-4954 | biostudies-arrayexpress
Project description:Introduction: Circulating microRNAs (miRNAs) exhibit remarkable stability and may serve as biomarkers in several clinical cancer settings. The aim of this study was to investigate changes in the levels of specific circulating miRNA following breast cancer surgery and evaluate whether these alterations were also observed in an independent data set. Methods: Global miRNA analysis was performed on prospectively collected serum samples from 24 post-menopausal women with estrogen receptor-positive early-stage breast cancer before surgery and 3 weeks after tumor resection using global LNA-based quantitative real-time PCR (qPCR). Results: Numbers of specific miRNAs detected in the samples ranged from 142 to 161, with 107 miRNAs detectable in all samples. After correction for multiple comparisons, 3 circulating miRNAs (miR-338-3p, miR-223 and miR-148a) exhibited significantly lower, and 1 miRNA (miR-107) higher levels in post-operative vs. pre-operative samples (p<0.05). No miRNAs were consistently undetectable in the post-operative samples compared to the pre-operative samples. Subsequently, our findings were compared to a dataset from a comparable patient population analyzed using similar study design and the same qPCR profiling platform, resulting in limited agreement. Conclusions: A panel of 4 circulating miRNAs exhibited significantly altered levels following radical resection of primary ER+ breast cancers in post-menopausal women. These specific miRNAs may be involved in tumorigenesis and could potentially be used to monitor whether all cancer cells have been removed at surgery and/or, subsequently, whether the patients develop recurrence.
2014-11-01 | GSE57661 | GEO
Project description:Introduction: Circulating microRNAs (miRNAs) exhibit remarkable stability and may serve as biomarkers in several clinical cancer settings. The aim of this study was to investigate changes in the levels of specific circulating miRNA following breast cancer surgery and evaluate whether these alterations were also observed in an independent data set. Methods: Global miRNA analysis was performed on prospectively collected serum samples from 24 post-menopausal women with estrogen receptor-positive early-stage breast cancer before surgery and 3 weeks after tumor resection using global LNA-based quantitative real-time PCR (qPCR). Results: Numbers of specific miRNAs detected in the samples ranged from 142 to 161, with 107 miRNAs detectable in all samples. After correction for multiple comparisons, 3 circulating miRNAs (miR-338-3p, miR-223 and miR-148a) exhibited significantly lower, and 1 miRNA (miR-107) higher levels in post-operative vs. pre-operative samples (p<0.05). No miRNAs were consistently undetectable in the post-operative samples compared to the pre-operative samples. Subsequently, our findings were compared to a dataset from a comparable patient population analyzed using similar study design and the same qPCR profiling platform, resulting in limited agreement. Conclusions: A panel of 4 circulating miRNAs exhibited significantly altered levels following radical resection of primary ER+ breast cancers in post-menopausal women. These specific miRNAs may be involved in tumorigenesis and could potentially be used to monitor whether all cancer cells have been removed at surgery and/or, subsequently, whether the patients develop recurrence. 48 serum samples were prospectively collected from 24 patients with early stage breast cancer before and after surgery at Odense University Hospital. Serum was prepared within one hour of sample collection after centrifugation (2000 x g; 10 min at 20 M-BM-:C) and immediately stored at -80 M-BM-:C.
2014-11-01 | E-GEOD-57661 | biostudies-arrayexpress
Project description:During microRNA-biogenesis, stem-loop structured precursors are processed in two endonucleolytic steps, giving rise to mature microRNAs (miRNAs). This process is not only regulated at the level of transcription but also posttranscriptionally by RNA-binding proteins (RBPs). Here we have used a proteomics-based pull-down approach to map and characterize the interactome of 72 pre-miRNAs in eleven cell lines. We identify over 150 RBPs that interact specifically with distinct pre-miRNAs. To demonstrate their functional relevance, we used RNAi knockdown and CRISPR/Cas-mediated knockout experiments and analyzed changes in miRNA levels. Indeed, a large number of the investigated candidates have effects on miRNA-processing under our experimental conditions, including several C3H-zinc-finger proteins. Ultimately, we show that TRIM71/Lin41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. In summary, we provide an extended database of RBPs that interact with pre-miRNAs in different cell types helping to elucidate posttranscriptional regulation of miRNA biogenesis on a global scale.
2017-04-26 | PXD004193 | Pride
Project description:We aimed to identify urinary exosomal miRNAs associated with PCa metastasis and develop a non-invasive risk-scoring model for PCa metastasis in this study. MiRNA profiles were examined using the Taqman low-density miRNA array (TLDA). Megaplex reverse transcription reactions and pre-amplification reactions were performed to increase the quantity of cDNA for miRNA expression analysis using the Megaplex PreAmp Primers Human Pool A and TaqMan PreAmp Master Mix (Thermo Fisher Scientific). MiRNA expression was evaluated via the TLDA panel A v2.0 (Thermo Fisher Scientific). Raw data were processed using the QuantStudio Real-Time PCR Software (Thermo Fisher Scientific) to determine a cycle threshold (Ct) value for each miRNA.
2021-06-07 | GSE173094 | GEO
Project description:Recently, long oligonucleotide (60-70mer) microarrays for two-color experiments have been developed and are gaining widespread use. In addition, when there is limited availability of mRNA from tissue sources, RNA amplification can and is being used to produce sufficient quantities of cRNA for microarray hybridization. Taking advantage of the selective degradation of RNA under alkaline conditions, we have developed a method to “strip” glass-based oligonucleotide microarrays that use fluorescent RNA in the hybridization, while leaving the DNA oligonucleotide probes intact and usable for a second experiment. Replicate microarray experiments conducted using stripped arrays showed high reproducibility, however, we found that arrays could only be stripped and reused once without compromising data quality. The intraclass correlation (ICC) between a virgin array and a stripped array hybridized with the same sample showed a range of 0.90-0.98, which is comparable to the ICC of two virgin arrays hybridized with the same sample. Using this method, once stripped oligonucleotide microarrays are usable, reliable, and should help to reduce costs. Keywords = Agilent microarray Keywords = Stripped arrays Keywords = Replicate reproducibility Keywords: other
2005-02-23 | GSE1706 | GEO