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.
Highlights
That a large amount of sequencing data has been obtained by the Human Genome Project (HGP), the big subject is to understand various biological phenomena from the viewpoint of system biology
It has been technically challenging to generate expression profiles from single cells, especially bias, such as PCR-introduced bias, sequence-dependent bias and degradation-induced bias, which is always produced by technical limitations
The main technical novelty of this work is the combination of bead-supported cDNA library preparation and unbiased amplification of cDNA from single cells
Summary
That a large amount of sequencing data has been obtained by the Human Genome Project (HGP), the big subject is to understand various biological phenomena from the viewpoint of system biology. Due to technical limitations, most gene expression analyses are carried out with a large number of cells. They give averaged data, which may mask important information. The authors previously developed a method of combining qPCR and a bead-supported singlecell cDNA library [11] This method allows the repeated use of a whole-cDNA library for quantifying the expression of each gene. DNA chips [12] and next-generation DNA sequencers (e.g., the SOLiD system) [13] have been widely used for gene expression analysis of the entire mRNA in single cells, it requires global cDNA amplification (which frequently causes bias)
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