Non-biased and efficient global amplification of a single-cell cDNA library.
Non-biased and efficient global amplification of a single-cell cDNA library.
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DOI：
10.1093/nar/gkt965
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发表时间：
2014-01
影响因子：
14.9
通讯作者：
Kambara H
中科院分区：
文献类型：
--
作者：
Huang H;Goto M;Tsunoda H;Sun L;Taniguchi K;Matsunaga H;Kambara H
单细胞基因表达的分析有望更精确地了解生命系统的分子机制。大多数技术只允许研究有限数量的基因物种的表达。当进行cDNA扩增以分析更多基因时，经常报告扩增偏倚。一个无偏见的和有效的全球性的扩增方法，它使用一个单细胞的cDNA文库固定在珠子上，被开发用于分析整个基因表达的单细胞。从逆转录到cDNA扩增的每一步分析都进行了优化。通过去除降解过量的引物，防止了由于cDNA的消化引起的偏倚。由于影响每个后续反应效率的残留试剂可以通过洗涤珠粒去除，因此实现了cDNA的均匀和最大化扩增的条件。随机选择的8个基因的扩增率差异在1.5倍以内，这对于单细胞分析的大多数应用来说可以忽略不计。整体扩增从单个细胞（2-pg mRNA）中得到大量扩增的cDNA（>100 μg），并且该量足以用于下游分析。所提出的全局扩增方法用于定量分析多个cDNA靶（从几个拷贝到几千个拷贝）的转录比。
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.
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