Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14 Str,, 61-704 Poznan, Poland.
BMC Mol Biol. 2011 Apr 11;12:14. doi: 10.1186/1471-2199-12-14.
Numerous microRNAs (miRNAs) have heterogeneous ends resulting from imprecise cleavages by processing nucleases and from various non-templated nucleotide additions. The scale of miRNA end-heterogeneity is best shown by deep sequencing data revealing not only the major miRNA variants but also those that occur in only minute amounts and are unlikely to be of functional importance. All RNA interference (RNAi) technology reagents that are expressed and processed in cells are also exposed to the same machinery generating end-heterogeneity of the released short interfering RNAs (siRNAs) or miRNA mimetics.
In this study we have analyzed endogenous and exogenous RNAs in the range of 20-70 nt by high-resolution northern blotting. We have validated the results obtained with northern blotting by comparing them with data derived from miRNA deep sequencing; therefore we have demonstrated the usefulness of the northern blotting technique in the investigation of miRNA biogenesis, as well as in the characterization of RNAi technology reagents.
The conventional northern blotting enhanced to high resolution may be a useful adjunct to other miRNA discovery, detection and characterization methods. It provides quantitative data on distribution of major length variants of abundant endogenous miRNAs, as well as on length heterogeneity of RNAi technology reagents expressed in cells.
许多 microRNAs (miRNAs) 由于加工核酶的不精确切割以及各种非模板核苷酸的添加而具有不均匀的末端。miRNA 末端异质性的规模最好通过深度测序数据显示出来，这些数据不仅揭示了主要的 miRNA 变体，还揭示了那些仅以微量存在且不太可能具有功能重要性的变体。所有在细胞中表达和加工的 RNA 干扰 (RNAi) 技术试剂也都暴露在相同的产生释放的短干扰 RNA (siRNA) 或 miRNA 模拟物的末端异质性的机制下。
在这项研究中，我们通过高分辨率 northern 印迹分析了 20-70nt 范围内的内源性和外源性 RNA。我们通过将 northern 印迹的结果与 miRNA 深度测序数据进行比较来验证结果；因此，我们证明了 northern 印迹技术在 miRNA 生物发生研究以及 RNAi 技术试剂的表征中的有用性。
增强到高分辨率的常规 northern 印迹可能是其他 miRNA 发现、检测和表征方法的有用补充。它提供了关于丰富内源性 miRNAs 的主要长度变体分布以及细胞中表达的 RNAi 技术试剂的长度异质性的定量数据。
