IRB Barcelona, Spain
Expression profiling, the measurement of all transcripts of a cell, is currently the most comprehensive method to describe its physiological state. Given that accurate profiling methods currently available require RNA amounts found in thousands to millions of cells, many fields of biology working with specialized cell types cannot use these techniques because available cell numbers are limited. Currently available alternative methods for expression profiling from picograms of RNA or from very small cell populations lack a broad validation of results to provide accurate information about the measured transcripts. Except for a few highly experimental methods like Single-Molecule-Sequencing, every attempted measurement of more than a few different transcripts needs a pre-amplification of cDNA. Here we present Pico Amplification, a novel workflow of RNA isolation and cDNA amplification for cell populations as small as a few or even a single cell. Micrograms of cDNA are generated, suitable for analysis by qPCR, microarrays or sequencing. Expression profiling of RNAs diluted to picograms showed that Pico Amplification virtually did not alter measurements of differential expression. MAQC samples A and B (Universal Reference RNA and Human Brain RNA) were utilized to provide transcriptome wide evaluation using microarrays and for comparison to qPCR measurements for over 800 transcripts. RNA isolated from 10 cells provided after Pico Amplification virtually identical information about the entire population as standard protocols do from thousands or millions of cells. This held true across the entire transcriptome. RNA isolated from individual cells provided insight into the heterogeneity of seemingly homogenous cell populations. Hundreds of transcripts were found to be differentially expressed between individual cells. Pico Amplification provides micrograms of cDNA from very small cell populations or even individual cells, highly representative of the original transcriptome. This allows the application of virtually all standard methods of expression profiling to interrogate the transcriptional status of an individual cell.
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