Abstract
Comparison of the performance of three methods for amplifying single-cell amounts of RNA for use in expression profiling shows that PCT amplification is more reliable than linear amplification.
Highlights
Single-cell microarray expression profiling requires 108-109-fold amplification of the picogram amounts of total RNA typically found in eukaryotic cells
R18.2 Genome Biology 2006, Volume 7, Issue 3, Article R18 Subkhankulova and Livesey http://genomebiology.com/2006/7/3/R18 techniques are based on two different approaches: linear isothermal amplification by in vitro transcription (IVT) of the cDNA population into labeled complementary RNA, typically using T7 RNA polymerase [5,6], and PCR amplification of the entire population of cDNA following reverse transcription [7,8,9]
Experimental design and yields of amplified DNA/ complementary RNA (cRNA) The objective of this study was to compare amplification techniques and choose the most reliable method for single-cell expression profiling
Summary
Single-cell microarray expression profiling requires 108-109-fold amplification of the picogram amounts of total RNA typically found in eukaryotic cells. Amplified RNA (aRNA) samples have been shown to generate reproducible microarray data when compared with non-amplified mRNA and closely approximate original samples [13,14,15]. It has been found, that the resulting microarray data can vary depending on details of the amplification protocol, including the amount of starting material [13], whether antisense or sense RNA is produced [16], and the number of rounds of amplification performed. Time-dependent RNA degradation during IVT can introduce noise to the resulting microarray data [17]
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