A global single-cell cDNA amplification method for quantitative microarray analysis.
We describe here a protocol to faithfully amplify global cDNAs from single cells. The amplified cDNAs retain their sense-antisense orientation and can be easily applied to template preparation for quantitative high-density oligonucleotide microarray analyses. The amplification protocol comprises (1) lysis of a single cell in a tube without purification, (2) first-strand cDNA synthesis with the first primer tailed with oligo dT, (3) elimination of the unreacted first primer, (4) poly (dA) tailing of the cDNA, (5) second-strand cDNA synthesis with the second primer tailed with oligo dT, and (6) 20-cycle, directional PCR with the two primers. To prepare the template for the isothermal linear amplification with T7 RNA polymerase to synthesize labeled cRNAs for microarray hybridization, the promoter sequence is added to the cDNA with another round of PCR. The promoter-tagged cDNA is purified with gel electrophoresis and amplified with one final cycle of PCR.
Laboratory for Mammalian Germ Cell Biology, RIKEN Center for Developmental Biology, Kobe, Japan. kurimoto@cdb.riken.jp
Kazuki
K
Kurimoto
Mitinori
M
Saitou
D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis
D017931:DNA Primers; D018076:DNA, Complementary