Chum-RNA allows preparation of a high-quality cDNA library from a single-cell quantity of mRNA without PCR amplification.
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 10(5) 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 (K(m)) values are around 1 mM, allowing them to be activated in the presence of only small quantities of substrate.
Tougan
Takahiro
T
Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Okuzaki
Daisuke
D
Nojima
Hiroshi
H
D018076:DNA, Complementary / Q000096:biosynthesis; D012321:DNA-Directed RNA Polymerases; D020869:Gene Expression Profiling; D015723:Gene Library; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D014764:Viral Proteins
D018076:DNA, Complementary; D012333:RNA, Messenger; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases