Inconsistent results with RNA quantification at A260/280 - AND problems with recoveries using Qiagen columns (Feb/07/2006 )
Can anyone help me troubleshoot?
I isolated RNA recently using the TRIzol procedure from plated cells in a 60 mm dish. The procedure went fine without any mistakes. I resuspended the RNA in 50 µL 1X TE and the readings from the spec showed me that I had about 4 µg/µL (I made 1:50 and 1:100 dilutions in 1X TE for the spectrophotometer).
Next, I used those calculations to add 45 µg RNA to Qiagen RNeasy MinElute Spin Columns. That procedure also went smoothly (as far as I know). But when I read the eluate at A260/280, I had recovered 0.04 µg/µL and less than 1 µg total!! (The readings were barely above background, so I don't even know if I really had anything in there.)
I went back to the original samples (pre-purification) and read them again at A260/280, and this time, I found 1.6 µg/µl - very different than the first readings.
Why did I get such inconsistent results from the original samples? And even if my originals were 1.6 µg/µL, I still should have recovered SOMETHING using the clean-up columns. Any clues?
Thanks a lot!
what was the goal of this second purif?...
for measuring accurate OD of your RNA, denature it 55° 10' and do the reading. After that thawing you may have not completely denature the RNA, which drives a reduced OD value.
In general, i believe that a lot of material is lost in columns. What can i suppose is that column wasn't enough dried and/or you didn't wait before elution-step-spin.
Hi Fred, thanks for your reply.
The goal of the column purification was to remove potential inhibitors remaining from the TRIzol isolation, such as phenol.
I did not know about the denaturing step! Thanks for that tip. Also, I think I saw somewhere on the boards that a 2nd spin before elution should be done - I didn't try that either.
make fresh nuclease-free TE just in case
the second spin is important. You get nearly 40µl of ethanol
and i let the column dry 15' rt minimum.
on trizol paper, you have a tip for preventing degradation (but depends of your goal) : you can at final step resuspend in formamide. But for RT PCR assays that imples a ethanol precipitation...
If you do northerns, this is fine.
Great, thanks guys.
Aimikins, great idea - I was using a 15-mL aliquot of 1X TE that I had on the bench. I will throw that tube out and get fresh TE.
Fred, I am doing RT PCR and would prefer to stick with 1X TE, but thanks for the idea. As for the 2nd spin, you are talking about the open-cap spin right before elution, is that right? So, you do that for another 5 minutes, and then let the tubes sit for 15 minutes (caps open or closed?), and then elute?
I have same exprience with columns. We use the Ambion Micro RNAqueous. It is just not consistent. But on gel after my PCR the band looks just fine (equal loading) so I just stick to this method.
For RTPCR, don't resuspend in formamide, you'll loose RNA when repelleting
As second spin i'm talking after the wash step, first spin and the second one (so before elution) is done cap closed for 1' at 10000g (more if your columns can, but 10 000 is enough in most cases)
then i transfer the column to an eppi and both caps are open .
Alternative used by a colleague for heating the column : she cuts the bottom of an eppi, insert the column in it and put it on the thermoblock (eppi is in this case only an "adaptator".