Low yield for small RNA isolation - (Jan/07/2013 )
I'm a newbie to the field of RNA.
I'm currently using phenol:chloroform:isoamylalcohol (25:24:1) for extracting RNA and subsequenty purified RNA (total and small RNA) using Purelink miRNA kits. What I had done as suggested by the kit was that, I subjected the crude RNA extracts to two column purification: the first by the addition of 35% ethanol to RNA (RNA species bound to column1 are large RNA species), the second by taking the eluate from first column and adding another 35% ethanol (final 70% ethanol), then passing the mixture to a second column (RNA species bound to column2 are small RNA species). Elution is done with 40ul RNase-free water.
However, I tried a couple of times and have gotten low yield of RNA (both total and small RNA). My source of RNA is from E. coli.
Have anyone tried using the kits and have problems like mine? Would like to hear any advices.
The gold standard to purify small RNA is using TRIzol/TRI Reagent. Zymo Research has a product called Direct-zol that allows you purify RNA using TRI Reagent directly on a spin column, without the need for phase separation or alcohol precipitation steps. Check it out here: http://www.zymoresearch.com/rna-purification/total-rna-purification/samples-in-tri-reagent-trizol-etc/direct-zol-rna-miniprep.
(Disclosure: I am an employee of Zymo Research, but Direct-zol really is an amazing product!)