I am trying to extract RNA from Chlamydomonas (Cw92) using traditional
Trizol method. My RNA yield comes up really good around 900 to 1000 ng/ul.
Even the 260/280 and 260/230 ratio’s are close to 2 or 2.2. I am using this
method from 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 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 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
looks fine on same machine. Now I am confuse, what I am doing wrong? Why I
am getting low RIN numbers in chlamy samples? Does anybody have any
suggestion to improve RIN number or RNA extraction protocol
Thanks in advance.
Dr. Amit Bajhaiya (Post-Doctoral Fellow)
Professor. Göran Samuelsson's lab
SE-901 87 Umeå
Tel.: +46 727717477
Email: amit.bajhaiya from umu.se