Wadhwa R, Duncan E, Kaul S C, Reddel R R
Children's Medical Research Institute, Sydney, Australia.
Mol Biotechnol. 1996 Oct;6(2):213-7. doi: 10.1007/BF02740775.
A reverse Northern analysis that effectively eliminates the false positives isolated from differential display of mRNAs (DD) is demonstrated. Preparation of probe by one-step labeling in reverse transcription reaction is found to be more effective and specific as compared to the preparation of probe by random priming of reverse transcribed cDNA pool. Reverse Northern assay of DNA fragments isolated from DD prior to their cloning into plasmid, analysis of multiple fragments on single slot blot, and requirement of RNA only in small amounts as compared to conventional Northern makes the protocol quick, effective, and economic.
本文展示了一种反向Northern分析方法,该方法能有效消除从mRNA差异显示(DD)中分离出的假阳性结果。与通过随机引物反转录cDNA文库制备探针相比,在反转录反应中通过一步标记制备探针更有效且特异。在将从DD中分离出的DNA片段克隆到质粒之前对其进行反向Northern分析、在单槽印迹上分析多个片段以及与传统Northern相比仅需少量RNA,使得该方案快速、有效且经济。