Department of Computational Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA.
Nucleic Acids Res. 2010 Apr;38(7):e98. doi: 10.1093/nar/gkp1235. Epub 2010 Jan 15.
The continuing discoveries of potentially active small RNAs at an unprecedented rate using high-throughput sequencing have raised the need for methods that can reliably detect and quantitate the expression levels of small RNAs. Currently, northern blot is the most widely used method for validating small RNAs that are identified by methods such as high-throughput sequencing. We describe a new northern blot-based protocol (LED) for small RNA (approximately 15-40 bases) detection using digoxigenin (DIG)-labeled oligonucleotide probes containing locked nucleic acids (LNA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide for cross-linking the RNA to the membrane. LED generates clearly visible signals for RNA amounts as low as 0.05 fmol. This method requires as little as a few seconds of membrane exposure to outperform the signal intensity using overnight exposure of isotope-based methods, corresponding to approximately 1000-fold improvement in exposure-time. In contrast to commonly used radioisotope-based methods, which require freshly prepared and hazardous probes, LED probes can be stored for at least 6 months, facilitate faster and more cost-effective experiments, and are more environmentally friendly. A detailed protocol of LED is provided in the Supplementary Data.
利用高通量测序以空前的速度不断发现潜在的活性小 RNA,这就需要开发能够可靠检测和定量小 RNA 表达水平的方法。目前,Northern blot 是最广泛用于验证通过高通量测序等方法鉴定的小 RNA 的方法。我们描述了一种新的 Northern blot 方法(LED),用于使用包含锁核酸(LNA)的 DIG 标记寡核苷酸探针检测小 RNA(约 15-40 个碱基),并用 1-乙基-3-(3-二甲基氨基丙基)碳二亚胺将 RNA 交联到膜上。LED 可以清晰地检测到低至 0.05 fmol 的 RNA 量。与基于同位素的过夜曝光方法相比,这种方法只需几秒钟的膜曝光时间即可获得更强的信号强度,曝光时间的改善约为 1000 倍。与常用的基于放射性同位素的方法不同,LED 探针可以储存至少 6 个月,便于进行更快、更具成本效益的实验,并且更加环保。详细的 LED 方案见补充数据。