Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of mouse samples subject to two different amplification methods for nanogram amounts of total rna to compare amplification methods

ABSTRACT: 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 One Round of amplification , Two round of amplification and RiboSPIA are listed here. All Hybridisation were performed using Affymetrix Mouse 430-2 gene chips. Data were all scaled to 500 and scaling factor average was 3.6 +/- 0.9, background intensities 46 +/- 5 , noise 2.9 +/- 0.6, rawQ 1.5 +/- 0.2

ORGANISM(S): Mus musculus

SUBMITTER: Philine Wangemann

PROVIDER: E-GEOD-1435 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Microarray-based comparison of three amplification methods for nanogram amounts of total RNA.

Singh Ruchira R Maganti Rajanikanth J RJ Jabba Sairam V SV Wang Martin M Deng Glenn G Heath Joe Don JD Kurn Nurith N Wangemann Philine P

American journal of physiology. Cell physiology 20041221 5

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 o ...[more]

PMID: 15613496

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Project description:Background: DNA microarrays provide a powerful method for global analysis of gene expression. The application of this technology to specific cell types and tissues, however, is typically limited by small amounts of available mRNA, thereby necessitating amplification. Here we compare microarray results obtained with two different methods of RNA amplification to profile gene expression in the C. elegans larval nervous system. Results: We used the mRNA-tagging strategy to isolate transcripts specifically from C. elegans larval neurons. The WT-Ovation Pico System (WT-Pico) was used to amplify 2 ng of Pan-neural RNA to produce labeled cDNA for microarray analysis. These WT-Pico-derived data were compared to microarray results obtained with a labeled aRNA target generated by two rounds of In Vitro Transcription (IVT) of 25 ng of Pan-neural RNA. WT-Pico results in a higher fraction of Present calls than IVT, a finding consistent with the proposal that DNA-DNA hybridization results in lower mismatch signals than the RNA-DNA heteroduplexes produced by IVT amplification. Microarray data sets from these samples were compared to a Reference profile of all larval cells to identify transcripts with elevated expression in neurons. These results were validated by the high proportion of known neuron-expressed genes detected in these profiles and by promoter-GFP constructs for previously uncharacterized genes in these data sets. Together, the IVT and WT-Pico methods identified 2,173 unique neuron-enriched transcripts. Only about half of these transcripts (1,044), however, are detected as enriched by both IVT and WT-Pico amplification. Conclusion: We show that two different methods of RNA amplification, IVT and WT-Pico, produce valid microarray profiles of gene expression in the C. elegans larval nervous system with a low rate of false positives. However, our results also show that each method of RNA amplification detects a unique subset of bona fide neural-enriched transcripts and thus a wider array of authentic neural genes are identified by the combination of these data sets than by the microarray profiles obtained with either method of RNA amplification alone. With its relative ease of implementation and greater sensitivity, WT-Pico is the preferred method of amplification for cases in which sample RNA is limiting. Keywords: expression profile

Dataset Information

Transcription profiling of mouse samples subject to two different amplification methods for nanogram amounts of total rna to compare amplification methods

Publications

Microarray-based comparison of three amplification methods for nanogram amounts of total RNA.

Similar Datasets

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