Broitman-Maduro Gina, Maduro Morris F
Department of Biology, University of California at Riverside, Riverside, California, USA.
Methods Cell Biol. 2011;106:253-70. doi: 10.1016/B978-0-12-544172-8.00009-8.
Detection of transcripts in situ is a rapid means by which gene expression can be characterized in many systems. In the nematode, Caenorhabditis elegans, the ease with which transgenics can be made and the general reliability of reporter fusion expression patterns, have made this technique comparatively less popular than in other systems. There are, however, still applications in which in situ hybridization is desired, such as for maternally expressed genes, or in related species without established transgene methods. The most frequently used method of in situ hybridization uses DNA probes and formaldehyde fixation. A newer approach that permits single-transcript detection has been reported and will not be described here (Raj and Tyagi, 2010). Rather, we describe an alternative protocol that uses RNA probes with a different fixative. This approach has been applied to C. elegans and related nematodes, providing reliable, sensitive detection of endogenous transcripts.
原位检测转录本是一种快速的方法，通过它可以在许多系统中对基因表达进行表征。在秀丽隐杆线虫中，制作转基因的简便性以及报告基因融合表达模式的总体可靠性，使得这项技术相比其他系统而言不太受欢迎。然而，仍然存在一些需要原位杂交的应用，例如对于母源表达的基因，或者在没有成熟转基因方法的相关物种中。最常用的原位杂交方法使用DNA探针和甲醛固定。已经报道了一种允许单转录本检测的新方法，这里将不做描述（Raj和Tyagi，2010）。相反，我们描述一种使用RNA探针和不同固定剂的替代方案。这种方法已应用于秀丽隐杆线虫和相关线虫，能够对内源转录本进行可靠、灵敏的检测。
