title: Non-biased and efficient global amplification of a single-cell cDNA library.
abstract: Analysis of single-cell gene expression promises a more precise understanding of molecular mechanisms of a living system. Most techniques only allow studies of the expressions for limited numbers of gene species. When amplification of cDNA was carried out for analysing more genes, amplification biases were frequently reported. A non-biased and efficient global-amplification method, which uses a single-cell cDNA library immobilized on beads, was developed for analysing entire gene expressions for single cells. Every step in this analysis from reverse transcription to cDNA amplification was optimized. By removing degrading excess primers, the bias due to the digestion of cDNA was prevented. Since the residual reagents, which affect the efficiency of each subsequent reaction, could be removed by washing beads, the conditions for uniform and maximized amplification of cDNAs were achieved. The differences in the amplification rates for randomly selected eight genes were within 1.5-folds, which could be negligible for most of the applications of single-cell analysis. The global amplification gives a large amount of amplified cDNA (>100 μg) from a single cell (2-pg mRNA), and that amount is enough for downstream analysis. The proposed global-amplification method was used to analyse transcript ratios of multiple cDNA targets (from several copies to several thousand copies) quantitatively.
authors.affiliation: Biosystems Research Department, Hitachi Central Research Laboratory, 1-280 Higashi-koigakubo, Kokubunji-shi, Tokyo 185-8601, Japan and Maternity Department, First Affiliated Hospital of Nanjing Medical University, Guangzhou Road 300, Nanjing 210029, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Huang
authors.forename: Mari
authors.initials: M
authors.lastname: Goto
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tsunoda
authors.forename: Lizhou
authors.initials: L
authors.lastname: Sun
authors.forename: Kiyomi
authors.initials: K
authors.lastname: Taniguchi
authors.forename: Hiroko
authors.initials: H
authors.lastname: Matsunaga
authors.forename: Hideki
authors.initials: H
authors.lastname: Kambara
mesh_terms: D002460:Cell Line; D017931:DNA Primers; D020869:Gene Expression Profiling; D015723:Gene Library; D007202:Indicators and Reagents; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D059010:Single-Cell Analysis
chemical_list: D017931:DNA Primers; D007202:Indicators and Reagents