What is the best method to measure miRNA levels? - (Apr/07/2007 )
There appears to be three good methods for assessing the levels of miRNAs: 1. northern analysis, 2. RNase protection assay, and 3. real-time PCR (TaqMan). The real-time approach seems to be the easiest but not everyone is using it, so I figure it must be either, very expensive or problematic. I would like to measure specific mouse and human miRNAs from tissue and cell lines so RNA quantity is not limited. I would appreciate any helpful tips as to which method would be best. Thanks in advance.
I would say the best and accurate method of detecting mature miRNA is Northern blotting, very straightforward, but tedious, tricky and radioactive. Taqman method is simpler but costly. Both methods have been widely used. Choice is made upon one’s preference. If money is not a problem, I will try Taqman first.
If you are going to assess many miRNAs at the same time, miRNA microarray is the choice.
The first step of isolating RNA and enriching small RNA is critical.
Hope that helps.
I’d prefer PCR based approach, especially polyA tailing based RT-PCR, since you can prepare it by yourselves in lab, even though there are already commercial kits available.
As to Northern blot, it is labor intensive and harmful (need radioactive reagents), it is also not sensitive and specific for the tiny size miRNA. LNA probe might increase the sensitivity and specificity, but very expensive…