36. QUANTITATIVE TRANSCRIPTOMICS USING DESIGNED PRIMER-BASED AMPLIFICATION

Department: Bioengineering
Research Institute Affiliation: Graduate Program in Bioinformatics
Faculty Advisor(s): Shankar Subramaniam | Vineet Bafna | Shyni Varghese

Primary Student
Name: Vipul Bhargava
Email: vibharga@ucsd.edu
Phone: 858-822-0960
Grad Year: 2012

Abstract
Quantification of low abundant transcripts from limited amounts of starting material has remained a challenge for RNA-seq at current sequencing depths. Here, we describe an informatics-based strategy that uses a defined set of heptamer primers to amplify the majority of transcripts expressed at moderate to low levels while preserving their relative abundance. Our strategy reproducibly yields high levels of amplification necessary for sequencing-library generation from low starting material and offers a dynamic range of over five orders of magnitude in RNA concentrations. Our method shows potential for selective amplification of transcripts of interest enabling better quantitation and multiplexing for increased throughput and cost effectiveness. We applied this approach to study cell lineage segregation in embryonic stem cell cultures, which models early mammalian embryogenesis. The amplification strategy revealed novel sets of low abundance transcripts, some corresponding to the identity of cellular progeny before they arise, reflecting the specification of cell fate prior to actual germ layer segregation.

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