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?