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Series GSE5130 Query DataSets for GSE5130
Status Public on Jun 23, 2006
Title Accurate and precise transcriptional profiles from 50 pg of total RNA or 100 flow-sorted primary lymphocytes
Organism Mus musculus
Experiment type Expression profiling by array
Summary 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
 
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
 
Contributor(s) Shearstone JR, Allaire NE, Campos-Rivera J, Rao S, Perrin S
Citation(s) 16624518
Submission date Jun 22, 2006
Last update date Jan 08, 2019
Contact name Jeff Shearstone
E-mail(s) jshearstone@acetylon.com
Phone 617-245-1321
Organization name Acetylon Pharmaceuticals
Department Biology
Street address 70 Fargo Street
City Boston
State/province MA
ZIP/Postal code 02210
Country USA
 
Platforms (1)
GPL339 [MOE430A] Affymetrix Mouse Expression 430A Array
Samples (103)
GSM115822 Mouse Universal Total RNA 500 ng AFFX2rd Labeling Replicate 1
GSM115823 Mouse Universal Total RNA 500 ng AFFX2rd Labeling Replicate 2
GSM115824 Mouse Universal Total RNA 50 ng AFFX2rd Labeling Replicate 1
Relations
BioProject PRJNA96635

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Supplementary file Size Download File type/resource
GSE5130_RAW.tar 305.3 Mb (http)(custom) TAR (of CEL)

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