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Accurate and precise transcriptional profiles from 50 pg of total RNA or 100 flow-sorted primary lymphocytes

ABSTRACT: We have developed a total RNA amplification and labeling strategy for use with Affymetrix GeneChips. Our protocol, which we denote BIIB, employs two rounds of linear T7 amplification followed by Klenow labeling to generate a biotinylated cDNA. In benchmarking studies using a titration of mouse universal total RNA, BIIB outperformed commercially available kits in terms of sensitivity, accuracy, and amplified target length, while providing equivalent results for technical reproducibility. BIIB maintained 50 and 44% present calls from 100 and 50 pg of total RNA, respectively. Inter- and intrasample precision studies indicated that BIIB produces an unbiased and complete expression profile within a range of 5 ng to 50 pg of starting total RNA. From a panel of spiked exogenous transcripts, we established the BIIB linear detection limit to be 20 absolute copies. Additionally, we demonstrate that BIIB is sensitive enough to detect the stochastic events inherent in a highly diluted sample. Using RNA isolated from whole tissues, we further validated BIIB accuracy and precision by comparison of 224 expression ratios generated by quantitative real-time PCR. The utility of our method is ultimately illustrated by the detection of biologically expected trends in a T cell/B cell titration of 100 primary cells flow sorted from a healthy mouse spleen. Keywords: Development and validation of an amplification and labeling protocol for transcriptional profiling on Affymetrix GeneChips, cDNA target on Affymetrix Genechip, small or limiting total RNA samples

ORGANISM(S): Mus musculus

PROVIDER: GSE5130 | GEO | 2006/06/23

SECONDARY ACCESSION(S): PRJNA96635

REPOSITORIES: GEO

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Transcription profiling of mouse tissue samples to assess an RNA amplification and labelling methodology

Project description:We have developed a total RNA amplification and labeling strategy for use with Affymetrix GeneChips. Our protocol, which we denote BIIB, employs two rounds of linear T7 amplification followed by Klenow labeling to generate a biotinylated cDNA. In benchmarking studies using a titration of mouse universal total RNA, BIIB outperformed commercially available kits in terms of sensitivity, accuracy, and amplified target length, while providing equivalent results for technical reproducibility. BIIB maintained 50 and 44% present calls from 100 and 50 pg of total RNA, respectively. Inter- and intrasample precision studies indicated that BIIB produces an unbiased and complete expression profile within a range of 5 ng to 50 pg of starting total RNA. From a panel of spiked exogenous transcripts, we established the BIIB linear detection limit to be 20 absolute copies. Additionally, we demonstrate that BIIB is sensitive enough to detect the stochastic events inherent in a highly diluted sample. Using RNA isolated from whole tissues, we further validated BIIB accuracy and precision by comparison of 224 expression ratios generated by quantitative real-time PCR. The utility of our method is ultimately illustrated by the detection of biologically expected trends in a T cell/B cell titration of 100 primary cells flow sorted from a healthy mouse spleen. Experiment Overall Design: Various experimental designs were used as part of the validation process for this total RNA amplification and labeling protocol. 103 samples were analyzed on Affymetrix GeneChips as part of this GEO series. Details of the BIIB protocol and supporting experiments are published in Genomics. 2006 Jul;88(1):111-21

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Mus musculus

Project description:Accurate and precise transcriptional profiles from 50 pg of total RNA or 100 flow-sorted primary lymphocytes

| PRJNA96635 | ENA

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