MiRNA Microarray Profiling with Three Different Total RNA Prep Methods
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE11840 | GEO | 2008/08/19
SECONDARY ACCESSION(S): PRJNA109095
REPOSITORIES: GEO
Similar Datasets
Project description:We performed miRNA microarray profiling on samples prepared from two different cell lines by three widely-used total RNA sample prep methods. miRNA microarrays were manufactured by Agilent Technologies (Santa Clara, CA), and contain 20-40 features targeting each of 470 human miRNAs (Agilent design ID 016436). Sequences of the 470 miRNAs were obtained from the Sanger miRBase, release 9.1. Labeling and hybridization of total RNA samples were performed according to the manufacturer’s protocol. 100ng total RNA was used as input into the labeling reaction, and the entire reaction was hybridized to the array for 20 hours at 55°C. For total RNA preps, frozen cell pellets were resuspended in phosphate buffered saline and divided into equal aliquots of 5x106 (HeLa) or 1x107 (ZR-75-1) cells and refrozen. Individual aliquots were subsequently thawed just before use.
2008-08-19 | E-GEOD-11840 | biostudies-arrayexpress
Project description:BackgroundDetermining the expression levels of microRNAs (miRNAs) is of great interest to researchers in many areas of biology, given the significant roles these molecules play in cellular regulation. Two common methods for measuring miRNAs in a total RNA sample are microarrays and quantitative RT-PCR (qPCR). To understand the results of studies that use these two different techniques to measure miRNAs, it is important to understand how well the results of these two analysis methods correlate. Since both methods use total RNA as a starting material, it is also critical to understand how measurement of miRNAs might be affected by the particular method of total RNA preparation used.ResultsWe measured the expression of 470 human miRNAs in nine human tissues using Agilent microarrays, and compared these results to qPCR profiles of 61 miRNAs in the same tissues. Most expressed miRNAs (53/60) correlated well (R > 0.9) between the two methods. Using spiked-in synthetic miRNAs, we further examined the two miRNAs with the lowest correlations, and found the differences cannot be attributed to differential sensitivity of the two methods. We also tested three widely-used total RNA sample prep methods using miRNA microarrays. We found that while almost all miRNA levels correspond between the three methods, there were a few miRNAs whose levels consistently differed between the different prep techniques when measured by microarray analysis. These differences were corroborated by qPCR measurements.ConclusionThe correlations between Agilent miRNA microarray results and qPCR results are generally excellent, as are the correlations between different total RNA prep methods. However, there are a few miRNAs whose levels do not correlate between the microarray and qPCR measurements, or between different sample prep methods. Researchers should therefore take care when comparing results obtained using different analysis or sample preparation methods.
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Project description:Gene expression profiling using microarrays requires microgram amounts of RNA, which limits its direct application for the study of nanogram RNA samples obtained using microdissection, laser capture microscopy, or needle biopsy. A novel system based on Ribo-SPIA technology (RS, Ovation-Biotin amplification and labeling system) was recently introduced. The utility of the RS system, an optimized prototype system for picogram RNA samples (pRS), and two T7-based systems involving one or two rounds of amplification (One RA, Standard Protocol, or Two RA, Small Sample Prototcol, version II) were evaluated in the present study. Mouse kidney (MK) and mouse universal reference (MUR) RNA samples, 0.3 ng to 10 mug, were analyzed using high-density Affymetrix Mouse Genome 430 2.0 GeneChip arrays. Call concordance between replicates, correlations of signal intensity, signal intensity ratios, and minimal fold increase necessary for significance were determined. All systems amplified partially overlapping sets of genes with similar signal intensity correlations. pRS amplified the highest number of genes from 10-ng RNA samples. We detected 24 of 26 genes verified by RT-PCR in samples prepared using pRS. Two RA yielded somewhat higher call concordances than did RS and pRS (91.8% vs. 89.3% and 88.1%, respectively). Although all target preparation methods were suitable, pRS amplified the highest number of targets and was found to be suitable for amplification of as little as 0.3 ng of total RNA. In addition, RS and pRS were faster and simpler to use than the T7-based methods and resulted in the generation of cDNA, which is more stable than cRNA.
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Project description:Since microRNAs (miRNAs) have been associated with eye diseases, our study aims to profile ocular miRNA expression in normal human eye tissue using miRNA-Seq in order to provide a foundation for future disease research. Total RNAs were extracted from normal human ciliary body (CB) (n=7), cornea (n=7), and trabecular meshwork (TM) (n=7) samples using mirVana total RNA isolation kit. The sequencing library was prepared using the Illumina TruSeq Small RNA Sample Prep kit and was sequenced using Illumina MiSeq. Generated sequence reads were trimmed and aligned against human reference database using the Bowtie software, and only exact matches to mature miRNAs from miRBase were included. miRTarBase database was used to analyze the gene targets of the miRNAs in our tissues, and expression of a few selected miRNAs were validated using droplet digital PCR (ddPCR). We found 378 miRNAs expressed collectively in our samples, of which miR-143-3p, miR-184, miR-26a-5p, and miR-204-5p were most abundantly expressed. With the expression patterns and gene targets, we identified several uniquely expressed miRNAs and created a profile of miRNAs known to target genes associated with keratoconus and glaucoma. Using ddPCR, we were able to validate the expression profile created using miRNA-Seq. For the first time, we profiled miRNA expression in three human ocular tissues using miRNA-Seq, identifying many miRNAs that had not been previously reported in ocular tissue. Knowing the expression of miRNAs in non-diseased eye tissues could help elucidate miRNA changes that accompany diseases such as glaucoma and keratoconus.
2016-08-01 | GSE81254 | GEO
Project description:Two T7 based methods One round of Amplification (Affymetrix) and Two round of Amplification were compared to two Ribo-SPIA based systems, RiboSPIA and pico Ribo SPIA systems. Data for Pico-RiboSPIA are listed here. All Hybridisation were performed using Affymetrix Mouse 430-2 gene chips. Data were all scaled to 500. For 12 chips performed with pico Ribo SPIA the scaling factor average was 2.0 +/- 0.3, background intensities 74.4 +/- 12.7 , noise 3.9 +/- 0.6, rawQ 2.5 +/- 0.3 Keywords: ordered
2004-12-21 | GSE2019 | GEO
Project description:Full-length 16S-23S rRNA gene sequences from pre and post PrEP specimens
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Project description:Since microRNAs (miRNAs) have been associated with eye diseases, our study aims to profile ocular miRNA expression in normal human eye tissue using miRNA-Seq in order to provide a foundation for future disease research. Total RNAs were extracted from normal human ciliary body (CB) (n=7), cornea (n=7), and trabecular meshwork (TM) (n=7) samples using mirVana total RNA isolation kit. The sequencing library was prepared using the Illumina TruSeq Small RNA Sample Prep kit and was sequenced using Illumina MiSeq. Generated sequence reads were trimmed and aligned against human reference database using the Bowtie software, and only exact matches to mature miRNAs from miRBase were included. miRTarBase database was used to analyze the gene targets of the miRNAs in our tissues, and expression of a few selected miRNAs were validated using droplet digital PCR (ddPCR). We found 378 miRNAs expressed collectively in our samples, of which miR-143-3p, miR-184, miR-26a-5p, and miR-204-5p were most abundantly expressed. With the expression patterns and gene targets, we identified several uniquely expressed miRNAs and created a profile of miRNAs known to target genes associated with keratoconus and glaucoma. Using ddPCR, we were able to validate the expression profile created using miRNA-Seq. For the first time, we profiled miRNA expression in three human ocular tissues using miRNA-Seq, identifying many miRNAs that had not been previously reported in ocular tissue. Knowing the expression of miRNAs in non-diseased eye tissues could help elucidate miRNA changes that accompany diseases such as glaucoma and keratoconus. This study profiled the expression of miRNAs in normal human ciliary body (n=7), cornea (n=7), and trabecualr meshwork (n=7) tissue
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