Northern杂交检测家蚕microRNA的技术流程

Protocol of Northern Blotting Hybridization for MicroRNA Detection of Silkworm(Bombyx mori )

microRNA(miRNA)是一类长度约22个碱基、具有重要转录后调控作用的非编码RNA。Northern杂交方法曾被认为是检测miRNA的金标准,但是由于影响实验结果的因素很多,要高质量地检测到这些短小、特殊的RNA分子也并非易事。在建立检测家蚕miRNA的Northern杂交技术平台基础上,详细介绍了相关的检测技术流程,包括miRNA的分离纯化、探针标记和Northern杂交的全过程,总结了该技术流程的特点以及操作中须重视的环节。所述内容具有很强的可操作性和实用性,可供研究人员检测家蚕miRNA和其他小RNA参考。

MicroRNAs（miRNAs） have been identified as non-coding RNAs with the length about 22 nucleotides.They play important roles in post-transcriptional regulation.Although Northern blotting is regarded as the golden standard for detecting the presence and level of miRNAs,to detect the specific miRNA molecules efficiently is still difficult because of its complicated operational procedure.In this study,the protocol of Northern blotting was described in detail based on our previous reports and unique experiences in miRNA studies,including the isolation and purification of miRNA,the labeling of probes and more details involved in Northern blotting,the characteristics of Northern blotting and key steps to be noted.The elaborated method is with high maneuverability and actual application value,providing good references for those who work on miRNAs and other small RNAs of silkworm.