220-plex microRNA expression profile of a single cell
220-plex microRNA expression profile of a single cell
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DOI：
10.1038/nprot.2006.161
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Surani, M. Azim
中科院分区：
文献类型：
--
作者：
Tang, Fuchou;Hajkova, Petra;Surani, M. Azim
在这里，我们描述了一个协议的检测microRNA（miRNA）的表达谱的单细胞干环实时PCR，这是特定的成熟的miRNA。单细胞首先通过热处理裂解而不进一步纯化。然后，通过茎环引物将220种已知的miRNA逆转录成相应的cDNA。随后是初始PCR步骤以扩增cDNA并产生足够的材料以允许单独的多重检测。将稀释的初始PCR产物用作模板以通过实时PCR检查个体miRNA表达。这种敏感的技术允许从单个细胞中进行miRNA表达谱分析，并允许分析来自早期胚胎的少数细胞以及单个细胞（如干细胞）。当只有纳克量的稀有样品可用时，也可以使用它。该方案可在7d内完成。
Here we describe a protocol for the detection of the microRNA (miRNA) expression profile of a single cell by stem-looped real-time PCR, which is specific to mature miRNAs. A single cell is first lysed by heat treatment without further purification. Then, 220 known miRNAs are reverse transcribed into corresponding cDNAs by stem-looped primers. This is followed by an initial PCR step to amplify the cDNAs and generate enough material to permit separate multiplex detection. The diluted initial PCR product is used as a template to check individual miRNA expression by real-time PCR. This sensitive technique permits miRNA expression profiling from a single cell, and allows analysis of a few cells from early embryos as well as individual cells (such as stem cells). It can also be used when only nanogram amounts of rare samples are available. The protocol can be completed in 7 d.