Quantitative analysis of mRNA amplification by in vitro transcription

• DOI: 10.1093/nar/29.5.e29
• Publication Date: 2002-07-26T18:52:52Z
• Authors: L. R. Baugh
• Keywords: Transcription

Effective transcript profiling in animal systems requires isolation of homogenous tissue or cells followed by faithful mRNA amplification. Linear amplification based on cDNA synthesis and vitro transcription is reported to maintain representation levels, however, quantitative data demonstrating this as well a description inherent limitations lacking. We show that published protocols produce template-independent product addition amplifying real target thus reducing the specific activity final product. describe modified protocol minimizes generation can therefore generate desired microgram quantities message-derived material from 100 ng total RNA. Application second, nested round reduces required starting 2 Quantitative analysis these products Caenorhabditis elegans Affymetrix GeneChips shows does not reduce overall sensitivity has only minor effects fidelity.