Checking integrity of total RNA using gel? - (Oct/21/2005 )
I am isolating total RNA from mouse liver with Trizol but can not check the quality of my sample.
After finishing isolation, the concentration of RNA sample checked by Spec. was 2.5 mg/ml which is enough for me. But, when I run the sample in 1% native agarose gel, I cound see any band but broad smeared thing aroung the dye.
For the ger roading, the 4microliter of sample was mixed with 4 microl MOPS, 7 microl formaldehyde and 20 microL formaide;denatured for 15 min at 55oC; and then mixed with 4microL of roading dye containing EtBr.
What can be the problem?
look at techtips and troubleshooting for RNA purity
I suspect your RNA is degrading; is all your stuff nuclease-free?
also, what is your 260/280? although I really think it is a degradation issue, not purity
The ratio was over 1.9.
And I think I used all RNase free stuffs.
1.9 is respectable
I don't know what it could be, then. perhaps someone else has an alternate solution?
I can't think of what would account for a big, low-MW smear except for degradation.
Yes i agree to that..This looks like a degradation issue. I am really surprised by your high RNA concantration. I usually struggle to get such high RNA concentrations using Trizol method . But I guess its probably go to do with the number of cells that are used to isolate RNA.
Hw about the 260/230 ratio ..Is that good enough?
Do you use any concentration method to concentrate the RNA . Well that might be a reason for degradation. I am not sure .aimikins, please give yor comments.
Can we use some method to concentrate my RNA samples. We have a vacuum dryer but not one that is attached to a lyophilizer.
As far as concentration being a factor in degradation, I don't know if I can help you. I usually isolate RNA from a fairly small number of cells (I use a 24-well culture dish, ~70-80% confluent) and I want to get the most mileage out of my samples.
I usually prep RNA via a kit; when eluting from the column I use a high amount of elution buffer to increase my overall yield and so I have a very dilute sample. I then do an ETOH precip step o/n at -80, then ETOH wash and spin. this leads to the most yield for my protocol.
I have no experience with vacuum or lyophilization methods of concentrating. I get very little degradation in the first several weeks, but over time even when stored carefully my samples will begin to degrade. I usually do qPCR, so I try to turn them over the cDNA as quickly as possible, usually within about 1 day of prepping.
by the time I am finished prepping and concentrating my samples, I usually get about 0.6-0.8mg/mL. I would not be so concerned about the high concentration as I would be about the smearing on the gel.
But, I am no expert. I only know what works for me.
i think that small RNA are more representative of quality of RNA.
I run a denaturing 15% PA gel 7M urea, and you'll should see sharp bands on it. I've attached two pictures. Shorts RNA correspond to the first 5 left lanes on the "long" picture...
It's just the gel stained with EtBr 15'. I cant post the detailed protocol if you want.
This is my example for checking quality of RNA... you see that in agarose gel quality seems ok, but in the acrylamide gel, except fro strange migration (due to a bubble i hadn't saw below the gel) you see little smearing
shorts ok are (obviously except the 2nd lane) better extracted.