Preparation of a high-quality cDNA library from a single-cell quantity of mRNA using chum-RNA.

Abstract

Unlike exponential amplification using polymerase chain reaction (PCR), linear RNA amplification using T7 RNA polymerase is advantageous for genome-wide analysis of gene expression and for cDNA library preparation from single-cell quantities of RNA. However, the use of RNA polymerase requires a large amount of RNA, as the optimum concentration of the substrate (mRNA), or the Michaelis constant (K(m)), is one millionfold higher than the single-cell amount of mRNA. To circumvent this K(m) problem, we designed a small mRNA-like dummy molecule, termed chum-RNA, which can be easily removed after the completion of the reaction. Chum-RNA allowed the preparation of a high-quality cDNA library from single-cell quantities of RNA after four rounds of T7-based linear amplification, without using PCR amplification. The use of chum-RNA may also facilitate quantitative reverse-transcription (qRT)-PCR from small quantities of substrate.