4.12. Northern blots using DIG labelling
The primary advantage of using Northern blot analyses for identifying specific predicted RNA’s, versus a PCR-based method, comes in the ability to predict the size of the entire transcript that is targeted using standard gel size markers. This is key especially when transcripts are subjected to editing (splice variants or enzymatic cutting as for small RNAs) and editing must be validated using a technique other than PCR. In addition, since probe binding is more permissive of nucleotide changes, Northern blots can be used to verify transcripts that might have mutations at primer sites used for PCR. In addition, this approach has somewhat lower vulnerability to point mutations that might cause a specific primer pair to fail to amplify a predicted target. The disadvantage to using Northern blots versus a PCR method as above is in time and expense and in a somewhat reduced ability to quantify transcript abundance. The below protocol avoids the use of radio-isotopic nucleotides as probes.