RNA extraction from pooled polysome fractions

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RapaSL's picture
RNA extraction from pooled polysome fractions

 My experiment requires extracting high quality RNA (good enough for RNA sequencing) from pooled polysome fractions. The polysomes are separated on sucrose gradients (7-47%) and fractions pooled to make 3 pools. 

I extracted RNA from liver pools using phenol chloroform. Because heparin is included in the gradient fractions, I have to do a LiCl ppt, otherwise heparin inhibits sequencing and RT-PCR.

I see layer inversion with phenol only extraction step, is this normal? I just noticed this the last two times. Thankfully, when I use the lower layer, I can get an aqueos layer in the 2nd extraction using phenol:CHCl3. Also, in one pool, my RNA quality is very poor. 260:280 <1.6 and lots of background on the bioanalyzer run. Is there a way to clean up these preps after Phenol:CHCl3 extraction? I have about 1-3 ug of RNA in some pools and I don't want to loose any. Is there other better methods of RNA extraction from polysome fractions?