Nonisotopic detection of microRNA using digoxigenin labeled RNA probes
Nonisotopic detection of microRNA using digoxigenin labeled RNA probes
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DOI：
10.1016/j.mcp.2005.07.004
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发表时间：
2006-02-01
影响因子：
3.3
通讯作者：
Kajigaya, S
中科院分区：
文献类型：
--
作者：
Rarnkissoon, SH;Mainwaring, LA;Kajigaya, S
微小RNA（miRNAs）是一类重要的内源性、约22个核苷酸的小分子非编码调节性RNA，最近发现它们与各种组织的发育、细胞调节和癌症有关。在此我们报道一种使用3'-地高辛（DIG）标记的RNA寡核苷酸探针检测miRNA的非同位素Northern分析方法。使用从人白血病细胞系中提取的miRNA或总RNA组分进行Northern印迹分析，并将印迹分别与针对miR - 181、miR - 155或miR - 16的P - 32或DIG标记的RNA探针进行杂交。一种针对U6小核RNA的标记探针用作内参。在检测低至50ng的miRNA量时，使用DIG标记的RNA探针与使用P - 32标记的探针具有同等的灵敏度。能够使用非同位素方法并获得灵敏且可靠的结果，这为那些希望避免使用同位素的研究人员提供了优势。由爱思唯尔有限公司出版。
MicroRNAs (miRNAs) are an important class of endogenously derived, small similar to 22 nucleotide noncoding regulatory RNAs that have recently become implicated in development, cell regulation and cancers of various tissues. Here we report a nonisotopic Northern analysis method for miRNA detection using 3 '-diaoxi-enin (DIG)-labeled RNA oligo probes. Northern blot analysis was performed using miRNA or total RNA fractions extracted from human leukemic cell lines, and blots were hybridized with either P-32- or DIG-labeled RNA probe for miR-181, miR-155 or miR-16. A labeled probe for U6 small nuclear RNA served as an internal control. The use of DIG-labeled RNA probes was equally sensitive compared to P-32-labeled probes in detecting miRNA quantities as low as 50 ng. The ability to use nonisotopic methods and yet obtain sensitive and reliable results offers an advantage to investigators who prefer to avoid isotopes. Published by Elsevier Ltd.