Experience with mRNA isolation kits? - (Jan/04/2012 )
Does anyone have suggestions for mRNA isolation kits? What kits have you used and how was the quality?
Any advice is appreciated.
For total RNA isolation we have used RNeasy and it works great. Also used Promega total RNA extraction kit with far less yield. Not sure about kits for mRNA only but I hope this helps.
Typically trizol works well. 70-100mg of tissue will yield a LOT of mRNA.
RNaqueous kits from Ambion work beautifully if you have difficult tissues (especially high fat content ones). I have gotten great results from 2mg pieces of brain and from adipose, which doesnt have much RNA. There is a micro kit (1-10mg) and the regular kit with columns that go to 100mg of tissue. Homogenize then spin down any debris that might clog the filter. Generally the RNA is very pure but (nanodrop readings at 2 for both ratios) but sometimes there will be contamination/carryover, which is fixed with an overnight lithium chloride precipitation. For microarray (or anything you need super clean RNA for) do a DNAase treatment then LiCl precip (both come with the micro kit, LiCl comes with the regular kit).
I used combination TRIZOL with RNeasy, it works well, I get high concentration and purity. Just take the aqueus phase after tizol treatment, and then transfer it to new tube, wash by 1 x volume of 70% ethanol, and transfer to the collumn. And you can go on with the normal procedure of RNeasy. Just skip homogenize the tissue with RLT buffer.
I have used RNAzol too. It is cheaper that Trizol. (100 ml is 110$ if you buy it from following site or 150$ if you buy it from its distributors, but TRIzol is 233$).