I found it easier to separate the sizes without a column (yield issues, strangely enough).  If you use a product like TRIzol or RNAzol, you would use different concentrations of ethanol (or isopropanol) to pull out one size set and then the other.

Here's RNAzol's protocol:

https://www.mrcgene.com/wp-content/uploads/2017/04/RNAzolRTMarch2017.pdf

Thanks for sharing! Using differential alcohol concentrations without a column makes sense - I believe that is the principle behind Qiagen's column based kit. Any idea if it would be better to 1) isolate total RNA first then use half of the fraction for an large RNA  library and the other half for a small library or 2) purify separate small and large fractions from total RNA then sequence. 

Also, any idea if there is anything proprietary about RNAzolRT or can I just perform a trizol/chloroform extraction to get an aqueous phase then precipitate the small and large fractions w/ different %'s of ethanol according to the protocol. Also, any insight/experience on performing DNase digestion on these fractions? I assume you would need to DNase digestion both to avoid contamination for cDNA library prep. Thanks!

Hi China,

I know that the RNAzol is different from TRIzol.  That said, in TRIzol, the RNA is in the initial aqueous phase, so you should be able to pull the different sizes down with different EtOH/Isopropanol concentrations.

As for DNA digest, if you've good hands and don't disturb the interphase, you shouldn't need the DNAse step, but if any concerns on that front, make sure your DNAse is free of RNAse contamination!

For your first asked question, you ought be able to use the same fraction for both small and large RNA (so option 2)

--Matt