Quantitative PCR Analysis of DNA, RNAs, and Proteins in the Same Single Cell
Quantitative PCR Analysis of DNA, RNAs, and Proteins in the Same Single Cell
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DOI：
10.1373/clinchem.2012.191445
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发表时间：
2012-12-01
影响因子：
9.3
通讯作者：
Aman, Pierre
中科院分区：
文献类型：
--
作者：
Stahlberg, Anders;Thomsen, Christer;Aman, Pierre
背景：单细胞代表了所有生物体的基本单位。大多数研究都是在大细胞群上进行的，但了解细胞动力学和异质性需要单细胞分析。目前的单细胞分析方法一般只能检测一类analyses.METHODS：逆转录和邻位连接法与定量PCR相结合，用于定量任何组合的DNA，mRNA，microRNA（miRNA），非编码RNA（ncRNA），和蛋白质从同一个单细胞。该方法被用于瞬时转染的人类细胞，以确定质粒，其转录的mRNA，翻译的蛋白质和下游RNA targets.RESULTS的细胞内浓度：我们开发了一种全细胞裂解缓冲液释放未分级的DNA，RNA和蛋白质，不会降低任何可检测的分析物或抑制测定。定量DNA、mRNA、miRNA、ncRNA和蛋白质的动态范围、分析灵敏度和特异性显示精确到单细胞水平。相关性研究显示，质粒及其转录mRNA的细胞内浓度与翻译蛋白浓度仅中度相关（斯皮尔曼相关系数分别为0.37和0.31; P < 0.01）。此外，异位表达的基因影响的分析物和这个基因，这是相关的基因regulation.CONCLUSIONS：这种方法是兼容的大多数细胞采样方法，并产生输出相同的参数为所有测量的分析物，一个功能，便于比较数据分析。这种方法应该开辟了新的途径，在分子诊断的详细相关性研究的多个和不同类别的分析物在单细胞水平。(C)2012年美国临床化学协会
BACKGROUND: The single cell represents the basic unit of all organisms. Most investigations have been performed on large cell populations, but understanding cell dynamics and heterogeneity requires single-cell analysis. Current methods for single-cell analysis generally can detect only one class of analytes.METHODS: Reverse transcription and the proximity ligation assay were coupled with quantitative PCR and used to quantify any combination of DNA, mRNAs, microRNAs (miRNAs), noncoding RNAs (ncRNAs), and proteins from the same single cell. The method was used on transiently transfected human cells to determine the intracellular concentrations of plasmids, their transcribed mRNAs, translated proteins, and downstream RNA targets.RESULTS: We developed a whole-cell lysis buffer to release unfractionated DNA, RNA, and proteins that would not degrade any detectable analyte or inhibit the assay. The dynamic range, analytical sensitivity, and specificity for quantifying DNA, mRNAs, miRNAs, ncRNAs, and proteins were shown to be accurate down to the single-cell level. Correlation studies revealed that the intracellular concentrations of plasmids and their transcribed mRNAs were correlated only moderately with translated protein concentrations (Spearman correlation coefficient, 0.37 and 0.31, respectively; P < 0.01). In addition, an ectopically expressed gene affected the correlations between analytes and this gene, which is related to gene regulation.CONCLUSIONS: This method is compatible with most cell-sampling approaches, and generates output for the same parameter for all measured analytes, a feature facilitating comparative data analysis. This approach should open up new avenues in molecular diagnostics for detailed correlation studies of multiple and different classes of analytes at the single-cell level. (C) 2012 American Association for Clinical Chemistry