Improved quantitative real-time RT-PCR for expression profiling of individual cells
Improved quantitative real-time RT-PCR for expression profiling of individual cells
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DOI：
10.1093/nar/gnf088
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发表时间：
2002-09-01
影响因子：
14.9
通讯作者：
Liss, B
中科院分区：
文献类型：
--
作者：
Liss, B
实时定量聚合酶链式反应 (rtqPCR) 克服了传统、耗时的定量 PCR 策略的局限性，并在将 mRNA 逆转录 (RT) 为互补 DNA (cDNA) 后，逐渐成熟为量化基因表达水平的常规工具。单细胞分辨率的表达谱分析是非常理想的，特别是对于像大脑这样包含大量不同类型细胞的复杂组织来说。膜片钳技术允许在记录细胞活动后选择性收获单细胞细胞质。然而，cDNA 反应的组分，特别是逆转录酶本身，会显着抑制随后的 rtqPCR 扩增。我直接使用未稀释的单细胞 cDNA 反应混合物作为 rtqPCR 的模板，观察到 rtqPCR 的扩增动力学以非系统方式发生了显着改变。在这里，我描述了一种简单而强大的沉淀方案，适用于纯化单细胞 cDNA，可完全去除抑制性 RT 成分，而不会检测到 cDNA 的损失。这种改进的单细胞实时 RT-PCR 方案提供了一个强大的工具来量化单个细胞的差异基因表达，从而可以补充基于全局微阵列的表达分析策略。
The real-time quantitative polymerase chain reaction (rtqPCR) has overcome the limitations of conventional, time-consuming quantitative PCR strategies and is maturing into a routine tool to quantify gene expression levels, following reverse transcription (RT) of mRNA into complementary DNA (cDNA). Expression profiling with single-cell resolution is highly desirable, in particular for complex tissues like the brain that contain a large variety of different cell types in close proximity. The patch-clamp technique allows selective harvesting of single-cell cytoplasm after recording of cellular activity. However, components of the cDNA reaction, in particular the reverse transcriptase itself, significantly inhibit subsequent rtqPCR amplification. Using undiluted single-cell cDNA reaction mix directly as template for rtqPCR, I observed that the amplification kinetics of rtqPCRs were dramatically altered in a non-systematic fashion. Here, I describe a simple and robust precipitation protocol suitable for purification of single-cell cDNA that completely removes inhibitory RT components without detectable loss of cDNA. This improved single-cell real-time RT-PCR protocol provides a powerful tool to quantify differential gene expression of individual cells and thus could complement global microarray-based expression profiling strategies.