title: 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.
authors.affiliation: Department of Molecular Genetics and DNA-chip Development Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan. snj-0212@biken.osaka-u.ac.jp
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nojima
authors.forename: Takahiro
authors.initials: T
authors.lastname: Tougan
mesh_terms: D000818:Animals; D018076:DNA, Complementary / Q000032:analysis* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D018274:Electroporation / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling; D015723:Gene Library; D056726:Genetic Association Studies; D046228:Microarray Analysis; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D012260:Ribonucleases