Gene expression in individual cells: analysis using global single cell reverse transcription polymerase chain reaction (GSC RT-PCR)

Leslie H Brail , Anne Jang , Filio Billia , Norman N Iscove , Henry J Klamut , Richard P Hill
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引用次数: 47

Abstract

The determination of the gene expression pattern of single cells has important implications for many areas of cellular and developmental biology including lineage determination, identification of primitive stem cells and temporal gene expression patterns induced by changes in the cellular microenvironment. Global Single Cell Reverse Transcription-Polymerase Chain Reaction (GSC RT-PCR) enables the study of single cell gene expression patterns. Initial observations of significant heterogeneity among single cells derived from a population of cells prompted us to determine how much of this observed heterogeneity was due to the intrinsic variation within the method. In this paper we discuss the sensitivity of GSC RT-PCR for analysis of differences in gene expression between single cells and, in particular, detail the amount of variation generated by the method itself. We found that most of the intrinsic variation in the method occurred in the PCR step. The total variation induced by the method was in the range of 5 fold. While we have determined that there is a five fold methodological variation in GSC RT-PCR, any method which use its components (including generation of cDNAs for microarray analysis) is likely to be affected by such experimental variability, which could limit the interpretation of the resulting data.

单细胞基因表达:全球单细胞逆转录聚合酶链反应(GSC RT-PCR)分析
单细胞基因表达模式的确定对细胞和发育生物学的许多领域具有重要意义,包括谱系确定、原始干细胞鉴定和细胞微环境变化诱导的时间基因表达模式。全球单细胞逆转录聚合酶链反应(GSC RT-PCR)使单细胞基因表达模式的研究。从细胞群中获得的单细胞之间的显著异质性的初步观察促使我们确定这种观察到的异质性有多少是由于方法内的内在变化。在本文中,我们讨论了GSC RT-PCR分析单细胞间基因表达差异的敏感性,特别是详细介绍了该方法本身产生的变异量。我们发现该方法的大部分内在变异发生在PCR步骤中。该方法引起的总变异在5倍范围内。虽然我们已经确定在GSC RT-PCR中存在五倍的方法差异,但任何使用其成分(包括生成用于微阵列分析的cdna)的方法都可能受到这种实验变异性的影响,这可能会限制对结果数据的解释。
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