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

Genomics
Jeffrey R ShearstoneSteven Perrin

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 biologicall...Continue Reading

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Citations

Jul 22, 2009·BMC Genomics·Julie E LangChristopher M Haqq
Mar 6, 2010·Bioinformatics·Jennifer ClarkeBertrand Clarke
Nov 26, 2015·Fertility and Sterility·Eleni MantikouMark de Jong
Jan 22, 2021·Journal of Nephrology·Harmandeep Kaur, Andrew Advani
Aug 11, 2010·Langmuir : the ACS Journal of Surfaces and Colloids·Stephanie E McCalla, Anubhav Tripathi

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