Best extraction method for miRNA

[wolfypita]

I was looking in the literature the methods that people use for extraction of miRNAs for sequencing. I found that people use the mirvana, the trizol, miRNesy....
In your view, which is the method that recover more miRNA? do you know if some of this methods has a bias for the different miRNAs?

Thank you

[FredBeaudoin]

I have the same question; indeed, at least one paper have done some work on this matter but it is about exosome... since I work with biopsies, the matter of quantity is not much of interest for me but the quality and the representativity of the RNA is more of my concern. For example, when we extract with RNeasy or Qiazol, we have ratio 260/280 and 260/230 >2.0.

If we look at the figure 6 of the paper, there are many peaks on the bioanalyser that are common but in fig 6a, 3 techniques (Trizol, Trizol+Clean-up, mirCURRY) have an over representation of small RNA from 100-150nt compared to miRNeasy and mirVANA... what is the best profile to hope for?

[mnkyboy]

I have had success with both mirVana and Trizol for mamallian cells and tissues.

Since we want do do whole transcriptome and small, we have just switched to the normal Trizol protocol because it keeps the small RNA fraction very well intact. If you only care about the small the mirVana works very well.

The exosome is a pain in our hands too, but that is on hold for us so we are not worrying about it now.

[FredBeaudoin]

how did you measure your 'success' I mean, what get me worried about is the representativity, the bias that can be introduce by the extraction/isolation technique...

[mnkyboy]

We get reproducibility from replicates, so we measure that as success.

Also publications. As for representativity do you mean numbers of small RNAs (diversity)?

Our systems are dominated by a small number of miRNAs highly expressed, but we see many others.

One thing to note is another reason we take the total RNA is because libraries will make cDNA for all sort of small RNAs so we find the library size selection criteria to be more important than the RNA extraction which we feel competent in.

I can make libraries that have are over 90% known miRNA, or I can change the size and get other interesting small non-coding RNAs.

[FredBeaudoin]

As for representativity do you mean numbers of small RNAs (diversity)?

I meant: if we want to compare ncRNA or miRNA to their specific mRNA abundance...

how do we make sure that there is no overepresentation induced by the technique (again look for the fig6 in the paper cited up)