Raj Arjun, Tyagi Sanjay
Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Methods Enzymol. 2010;472:365-86. doi: 10.1016/S0076-6879(10)72004-8.
Measurements of gene expression within single cells have revealed startling variability otherwise hidden in bulk measurements. Here, we present an in situ hybridization method capable of detecting individual mRNA molecules, thus permitting the accurate quantification and localization of mRNA within fixed sample. Our in situ protocol involves probing the target mRNA using a series of singly labeled oligonucleotide probes. This method is simple to implement and is applicable to a variety of biological samples. We also discuss some aspects of image processing required for analyzing the resulting data.
对单细胞内基因表达的测量揭示了原本隐藏在大量测量中的惊人变异性。在此，我们提出一种能够检测单个mRNA分子的原位杂交方法，从而能够在固定样本中对mRNA进行准确的定量和定位。我们的原位实验方案包括使用一系列单标记寡核苷酸探针探测目标mRNA。该方法易于实施，适用于多种生物样本。我们还讨论了分析所得数据所需的图像处理的一些方面。
