Søe Martin Jensen, Dufva Martin, Holmstrøm Kim
Bioneer A/S, Kogle Allé 2, 2970, Hørsholm, Denmark.
Methods Mol Biol. 2014;1173:113-21. doi: 10.1007/978-1-4939-0931-5_10.
In situ hybridization is a powerful method to provide information about contextual distribution and cellular origin of nucleic acids, e.g., in formalin-fixed paraffin-embedded (FFPE) samples of tissue. Particularly the recently discovered classes of noncoding RNA (ncRNA) including endo-siRNAs and microRNAs require such a technique to enable their study and visualization in natural contexts, and in the last decade, many advances have been made, increasing our ability to specifically detect small ncRNAs. One of the key developments has been the demonstration of the superiority of using locked nucleic acid (LNA)-modified DNA probes for the detection of ncRNA in tissue. Here, we describe an alternative in situ hybridization protocol employing oligonucleotide probes consisting of combinations of LNA and 2´-O-methyl RNAs that under optimized hybridization buffer conditions can provide a highly sensitive assay performance with only 1 h hybridization time.
原位杂交是一种强大的方法，可用于提供有关核酸的背景分布和细胞起源的信息，例如在福尔马林固定石蜡包埋（FFPE）的组织样本中。特别是最近发现的包括内源性小干扰RNA（endo-siRNA）和微小RNA在内的非编码RNA（ncRNA）类别，需要这样一种技术来使其能够在自然环境中进行研究和可视化。在过去十年中，已经取得了许多进展，提高了我们特异性检测小ncRNA的能力。关键进展之一是证明了使用锁核酸（LNA）修饰的DNA探针检测组织中ncRNA的优越性。在这里，我们描述了一种替代的原位杂交方案，该方案采用由LNA和2'-O-甲基RNA组合而成的寡核苷酸探针，在优化的杂交缓冲液条件下，仅需1小时的杂交时间即可提供高度灵敏的检测性能。
