Hello Everyone,

I am trying to extract RNA from Chlamydomonas (Cw92) using the traditional Trizol method. My RNA yield comes up really good around 900 to 1000 ng/ul. Even the 260/280 and 260/230 ratios are close to 2 or 2.2. I am using this method for a long time and I always trust these ratios and yield given by Nano-drop. I know Nano-drop measures only nucleic acid and it cannot differentiate very well between RNA and DNA.As I am extracting this RNA for RNA sequencing, where

As I am extracting this RNA for RNA sequencing, where the integrity of RNA is important.  The problem, which I came across, is that I always get good RNA and Ratio’s but when I do Bioanalyzer with the same RNA, the RIN number come out to be between 2 or 3. The RNA with such a low RIN number can not be trusted for sequencing and qPCR.

Earlier I thought RNAase might be the main problem so I tried using beta-mercaptoethanol with Trizol but there was not much improvement in RIN number. I have also checked bioanalyzer machine with other samples and they look fine on the same machine.  Now I am confused, what I am doing wrong? Why am I getting low RIN numbers in chlamy samples? Does anybody have any suggestion to improve RIN number or RNA extraction protocol?

Thanks in advance.