Nucleic Acids Research Advance Access published online on July 4, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn420
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Methods Online |
Chum-RNA allows preparation of a high-quality cDNA library from a single-cell quantity of mRNA without PCR amplification
Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
*To whom correspondence should be addressed. Tel: +81 6 6875 3980; Fax: +81 6 6875 5192; Email: snj-0212{at}biken.osaka-u.ac.jp
Received March 18, 2008. Revised June 7, 2008. Accepted June 18, 2008.
Linear RNA amplification using T7 RNA polymerase is useful in genome-wide analysis of gene expression using DNA microarrays, but exponential amplification using polymerase chain reaction (PCR) is still required for cDNA library preparation from single-cell quantities of RNA. We have designed a small RNA molecule called chum-RNA that has enabled us to prepare a single-cell cDNA library after four rounds of T7-based linear amplification, without using PCR amplification. Chum-RNA drove cDNA synthesis from only 0.49 femtograms of mRNA (730 mRNA molecules) as a substrate, a quantity that corresponds to a minor population of mRNA molecules in a single mammalian cell. Analysis of the independent cDNA clone of this library (6.6 x 105 cfu) suggests that 30-fold RNA amplification occurred in each round of the amplification process. The size distribution and representation of mRNAs in the resulting one-cell cDNA library retained its similarity to that of the million-cell cDNA library. The use of chum-RNA might also facilitate reactions involving other DNA/RNA modifying enzymes whose Michaelis constant (Km) values are around 1 mM, allowing them to be activated in the presence of only small quantities of substrate.