Impact of RNA isolation protocols on RNA detection by Northern blotting.

We prepared total RNA from the Gram-positive soil bacterium Bacillus subtilis by different RNA extraction procedures to compare their suitability for Northern blot detection of tiny RNAs (~14-mers) or RNAs of intermediate size (100-200 nt) in terms of signal quality, intensity, and reproducibility. Our analysis included two hot phenol methods and two TRIzol extraction procedures. We found that signal intensity/detection sensitivity makes the key difference. Total RNAs prepared by the hot phenol method comprise the length spectrum from tRNAs to large ribosomal RNAs. Larger RNAs are less abundant in TRIzol preparations which instead enrich for RNAs of tRNA size and smaller. Thus, hot phenol methods are the choice for the detection of intermediate-sized and longer RNAs, whereas TRIzol extraction procedures are more suited for the detection of tiny RNAs.