QC in RNA extraction - Low A260/A230 ratio (Nov/12/2008 )
Actually, I´m setting up a protocol for RNA extraction from frozen blood. I use a column-based kit from Promega. Frozen blood is a hard sample to work due to the low RNA concentration in leucocytes and the presence of haemoglobin and membrane rests that often limits the amount of sample to 1 mL max.
My firsts experiments yielded RNA in good concentration and purity (measured as A260/A280 ratio, close to 2), but poor A260/A230 ratio. Low A260/A230 ratio indicates the presence of salts (like GITC used to get the lysate), proteins or other products in the RNA solution that may interfere in further process.
If I try to re-precipitate the RNA solution to remove salts, then the RNA concentration falls significantly.
If I repeat washes during RNA purification the RNA concentration falls as well.
It seems to be a thin limit between concentration and purity, so I hope someone helps me to reach that point.
Thank you in advance!
what is the downstream application for your RNA? cDNA synthesis? You could go on with a few samples and check whether this low ratio will affect your downstream application at all. We sometimes have low ratios too but never observed any problems with cDNA synthesis.
The main application of my RNA is QF-PCR. We want to quantify the expression of a few genes involved in Noonan Syndrome.
For this reason, I think not to be good idea to run the experiment with some products in my sample absorbing in UV range.
Thanks for your answer!