Várallyay Eva, Havelda Zoltán
Agricultural Biotechnology Center, Gödöllő, Hungary.
Methods Mol Biol. 2011;732:9-23. doi: 10.1007/978-1-61779-083-6_2.
MicroRNAs (miRNAs) are short, about 21 nucleotides in length, non-coding, regulatory RNA molecules representing a new layer in post-transcriptional gene expression regulation. Spatial and temporal analysis of miRNA accumulation by in situ analyses is the prerequisite of understanding the precise biological functions of miRNAs. Since miRNAs are very short molecules, their in situ analysis is technically demanding. Our method is based on the usage of highly sensitive LNA-modified oligonucleotide probes. LNA modification significantly enhances the sensitivity and specificity of miRNA detecting probes and provides relatively easy in situ miRNA detection. Here, we describe a protocol for this challenging technique step by step, in order to help every user to achieve success.
微小RNA（miRNA）是一类短的、长度约为21个核苷酸的非编码调节性RNA分子，代表了转录后基因表达调控的一个新层面。通过原位分析对miRNA积累进行时空分析是理解miRNA精确生物学功能的前提。由于miRNA是非常短的分子，其原位分析在技术上要求很高。我们的方法基于使用高灵敏度的锁核酸（LNA）修饰的寡核苷酸探针。LNA修饰显著提高了miRNA检测探针的灵敏度和特异性，并提供了相对简便的miRNA原位检测方法。在此，我们逐步描述这一具有挑战性的技术的实验方案，以帮助每个用户取得成功。
