Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis.
We describe here a protocol for the representative amplification of global mRNAs from typical single mammalian cells to provide a template for high-density oligonucleotide microarray analysis. A single cell is lysed in a tube without purification and first-strand cDNAs are synthesized using a poly(dT)-tailed primer. Unreacted primer is specifically eliminated by exonuclease treatment and second strands are generated with a second poly(dT)-tailed primer after poly(dA) tailing of the first-strand cDNAs. The cDNAs are split into four tubes, which are independently directionally amplified by PCR, and then recombined. The amplified products (approximately 100 ng) show superior representation and reproducibility of original gene expression, especially for genes expressed in more than 20 copies per cell, compared with those obtained by a conventional PCR protocol, and can effectively be used for quantitative PCR and EST analyses. The cDNAs are then subjected to another PCR amplification with primers bearing the T7 promoter sequence. The resultant cDNA products are gel purified, amplified by one final cycle and used for isothermal linear amplification by T7 RNA polymerase to synthesize cRNAs for microarray hybridization. This protocol yields cDNA templates sufficient for more than 80 microarray hybridizations from a single cell, and can be completed in 5-6 days.
Kurimoto
Kazuki
K
Laboratory for Mammalian Germ Cell Biology, Center for Developmental Biology, RIKEN Kobe Institute, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
Yabuta
Yukihiro
Y
Ohinata
Yasuhide
Y
Saitou
Mitinori
M
D000818:Animals; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D051379:Mice; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger