Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.
Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.
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DOI：
10.1021/ac500459p
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发表时间：
2014-03-18
影响因子：
7.4
通讯作者：
Fodor, Stephen P. A.
中科院分区：
文献类型：
--
作者：
Fu, Glenn K.;Wilhelmy, Julie;Stern, David;Fan, H. Christina;Fodor, Stephen P. A.
我们提出了一种新的方法，灵敏地检测和准确定量的信使核糖核酸（mRNA）基因转录单细胞。首先，整个mRNA群体在逆转录期间用分子条形码编码。扩增目标基因靶标后，通过测序对分子条形码进行计数或在简单的杂交检测器上进行评分，以揭示起始样品中的分子数量。由于测量的是绝对量，因此无需校准标准品，并且避免了许多相对定量挑战，如聚合酶链反应（PCR）偏倚。我们将该方法应用于微小样品量的基因表达分析，并证明了精确的测量灵敏度下降到亚单细胞水平。该方法是一种简单的单管终点检测方法，使用标准热循环仪和PCR试剂。获得准确和精确的测量，而不需要任何基于周期到周期强度的实时监测或物理划分为多个反应（例如，数字PCR）。此外，由于所有mRNA分子都用分子条形码编码，因此扩增可用于产生更多的材料用于多次测量，并且可对有限的样品进行技术重复。该方法特别适用于少量样品，如单细胞实验。细胞内容的数字编码保留了真实的丰度水平，并克服了放大引起的失真。
We present a new approach for the sensitive detection and accurate quantitation of messenger ribonucleic acid (mRNA) gene transcripts in single cells. First, the entire population of mRNAs is encoded with molecular barcodes during reverse transcription. After amplification of the gene targets of interest, molecular barcodes are counted by sequencing or scored on a simple hybridization detector to reveal the number of molecules in the starting sample. Since absolute quantities are measured, calibration to standards is unnecessary, and many of the relative quantitation challenges such as polymerase chain reaction (PCR) bias are avoided. We apply the method to gene expression analysis of minute sample quantities and demonstrate precise measurements with sensitivity down to sub single-cell levels. The method is an easy, single-tube, end point assay utilizing standard thermal cyclers and PCR reagents. Accurate and precise measurements are obtained without any need for cycle-to-cycle intensity-based real-time monitoring or physical partitioning into multiple reactions (e.g., digital PCR). Further, since all mRNA molecules are encoded with molecular barcodes, amplification can be used to generate more material for multiple measurements and technical replicates can be carried out on limited samples. The method is particularly useful for small sample quantities, such as single-cell experiments. Digital encoding of cellular content preserves true abundance levels and overcomes distortions introduced by amplification.
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影响因子：
46.9
作者：
通讯作者：
--
影响因子：
48
作者：
Wu, Angela R.;Neff, Norma F.;Kalisky, Tomer;Dalerba, Piero;Treutlein, Barbara;Rothenberg, Michael E.;Mburu, Francis M.;Mantalas, Gary L.;Sim, Sopheak;Clarke, Michael F.;Quake, Stephen R.
通讯作者：
Quake, Stephen R.
DOI：
10.1073/pnas.1118018109
发表时间：
2012-01-24
影响因子：
11.1
作者：
Shiroguchi, Katsuyuki;Jia, Tony Z.;Xie, X. Sunney
通讯作者：
Xie, X. Sunney
影响因子：
48
作者：
Picelli, Simone;Bjorklund, Asa K.;Sandberg, Rickard
通讯作者：
Sandberg, Rickard
DOI：
10.1073/pnas.1323732111
发表时间：
2014-02-04
影响因子：
11.1
作者：
Fu, Glenn K.;Xu, Weihong;Fodor, Stephen P. A.
通讯作者：
Fodor, Stephen P. A.