I'm new to this board and have been reading it for a long time, and I think you are doing a really good job.I decided to register myself, because I have problems regarding my RNA Isolation. I want to isolate RNA from cultured PBMCs (round about 10^6 or less cells).
When I started up with Trizol I had very bad 260/280 ratios (~1,3) but I got along with that and reached a point where it is between 1,7-2.0.
But now I'm faced with bad 260/230 ratios (between 0,1 and 0,7).
I did the isolation according to the TRIpure protocol.
I put the extinction curve from the nanodrop reader into the attachment, which consists of two isolations.
In the first Isolations I had much excess ethanol in my samples (15-20 microliters) which might cause the bad ratios.
Now I tried to optimize the progress in the second RNA isolation and discarded all supernatants always keeping sure that the pellet is in the eppi. I also could see the dried transparent pellet after the last ethanol washing step (75%).I diluted the pellet in 25 microliters nuclease free water and incubated it for 10 mins at 60°C and stored it at -80°C.Today I went to measure it in the nanodrop. I could see that there was almost none RNA left. I had a yield of 30ng/per microliter, normally I had values like 300-1000ng per microliter. Though the 230nm extinction was less regarding the other isolations, I have a very bad 260/230 ratio, because there is almost no extinction at 260nm.
Does anybody know why I'm having such bad ratios?Troubleshooting the second isolation: Don't the curves show that my contamination got less and that I somehow just lost my pellet? The pellet was at first slight whitish and after drying it got quite transparent and filamentous. If it wasn't RNA in my pellet, because the nanodrop doesn't show much extinction at 260nm, what was is then, regarding that the 230nm and 280nm extinction is much lower than in the first isolation.
Could it be, that the RNA just did not dilute in the nuclease free water, though i also heated it for 10 mins? I used four samples for the second isolation. I overdried the first sample for along 40 mins. I dried the last pellet for about 10 mins and there is no difference in results...
I'm getting quite frustrated with Trizol... I would be thankful for every advice,
best regards Alex
Edited by AlexanderA, 18 November 2010 - 01:14 PM.