I was wondering if people could share their method for what they do with their total RNA after initial extraction, the downstream application being qRT-PCR. It is almost never mentioned in the methods section of papers. Specifically: 1) Do you usually use any RNA clean-up kits or DNAse treatment either before or after getting the RNA concentration (if so, specific brands would be helpful) and 2) which of these do you use to determine RNA concentration and integrity: 1) NanoDrop 1000 2) Qubit 3) Bioanalyzer 2100, anything else? 3) Or do you typically run it out on a gel combined with one of the methods in #2?
Hi Beata, after total RNA extraction we use Qiagen’s QuantiTect RT Kit. It’s great because it has a built in gDNA removal step before the RT reaction.
To determine the RNA integrity we normally use the NanoDrop and then run the sample on a gel. I think using the Bioanalyzer is a bit overkill.
Thank you. Is the gDNA clean-up step before you determine the RNA concentration?
I’ve talked to a few people recently and seen a few forums, which suggest that the NanoDrop (at least the UV spec method) grossly underestimates the true RNA concentration because it cannot distinguish between DNA and RNA. So, I would imagine you would want to clean up your sample first, then get the concentration and to check for RNA integrity run on a gel before proceeding with cDNA synthesis?
I don’t think the Nanodrop is that far off, actually. I’ve got decently close concentrations (+/- 5-10ng/uL) when I’ve compared Nanodrop and Bioanalyzer readings, but it depends on how clean your sample is.
The order is as such: RNA extraction → Nanodrop/run on gel → gDNA/RT reaction.
You really should have trace gDNA present before you get to that point. If you want to be super careful and you’ve got the money, you can buy kits like the RNeasy kit from Qiagen. You get super clean RNA and it even has an optional on-column DNase treatment step.
I second Snug’s comments. We use a Trizol extraction followed by purification with the Qiagen RNEasy column and we use the on-column DNaseI digestion stop. In my experience, this produces extremely clean RNA. ie. in RT-PCR analysis with a no-RT control we see either no signal or Cqs in the late 30s or 40s. I also test reference genes to ensure that the reference is appropriate when I start a given experiment experiment by examining the std dev of the Cqs across all of my samples. This relies on accurate RNA concentration determination and accurate pipetting. Using the nanodrop to quantitate the RNA has definitely been accurate enough.
Thank you both. Some of my NanoDrop curves do not look right even though the 260/280 ratio shows the sample to be pure (over 2) and the RNA concentration seems high enough. I take it, the thing to do is to run it out on a gel to check the band integrity and then run it through the RNeasy mini kit. Afterwards, there is no need to check the concentration/purity again and it’s okay to proceed with cDNA synthesis. Did I get that right? Or do you check it on the NanoDrop an additional time after the RNeasy or similar clean-up step? (in which case we are ok to assume that the gDNA does not interfere with the concentration reading)
You should run it on the column after Trizol purification, as Jordan says, and then Nanodrop/run on gel (if necessary). The reason why you should quantify your RNA after the column is that the column won’t elute everything you load on to it. Most of it comes off, but some of the RNA will be retained on the column. I’ve never done a ‘test’ to see how much you actually do lose, but it just makes more sense to just measure after than before.