Effect of freeze thaw on low abundance targets

[Nopainnoscience]

I snap froze a series of cell pellets to extract RNA, perform RT, then qPCR. Some of the snap frozen cell pellets may have briefly thawed and refrozen due to an issue with our freezer. I was wondering if anyone knew what kind of effect this would have on the RNA, especially since some of the targets are in fairly low abundance and since some of the samples may not have thawed.

This is concerning to me because the genes we normalize to (cd79b, gapdh) are highly abundant relative to the target. Thus, if RNA was degraded, it could have a large effect on the target gene abundance, but relatively little effect on the normalization gene. Is this a valid assumption? This is especially troubling since the cells are from a double KO mouse that is hard to breed and we do not have any more currently. Any advice or experience with this issue would be greatly appreciated.

[Anais1979]

Heh, this is why studying the expression of low abundance gene is a very questionable issue.

You never know. There is always a bit of degradation going on, and thaws will only accelerate it.

I dont think anyone will give a definite answer here.

[danfive]

Originally Posted by Nopainnoscience

I snap froze a series of cell pellets to extract RNA, perform RT, then qPCR. Some of the snap frozen cell pellets may have briefly thawed and refrozen due to an issue with our freezer. I was wondering if anyone knew what kind of effect this would have on the RNA, especially since some of the targets are in fairly low abundance and since some of the samples may not have thawed.

This is concerning to me because the genes we normalize to (cd79b, gapdh) are highly abundant relative to the target. Thus, if RNA was degraded, it could have a large effect on the target gene abundance, but relatively little effect on the normalization gene. Is this a valid assumption? This is especially troubling since the cells are from a double KO mouse that is hard to breed and we do not have any more currently. Any advice or experience with this issue would be greatly appreciated.

RNA is generally considered to be unstable. Freezing is the best storage. But the best thing is to do the best extraction, like Trizol RNA extraction. Very tough for RNAse to survive that phenol:choloform:thiocyanate treatment, phase separation and alcohol washes. So your Trizol extracted RNA in RNase free tubes, buffer etc.. is going to be very stable (relatively speaking).

If you have a similar clean Rnase-free RNA, then the next factor for stability is concentration. A high concentration is best. After each freeze-thaw you lose 10% of pure rH protein to insolubility(aggregation); it is safe to assume a similar loss in RNA.

I've worked with low abundance targets in low RNA yields (that is using body fluids with little nucleic acid to begin with). Optimized RT-PCR (both 1-step and 2-step) worked fine no loss of signal between interday assays etc. Most likely due to the high efficiency from an optimized (RT)PCR.

So hedge your bets with:
1. Best RNA extraction method available
2. Keeping things RNase free
3. Optimized RT-PCR

For your housekeeping genes: GAPDH, b-actin etc are expressed constitutively as you know, and their PCR efficiency will be different from your low abundance target. I'd care more about PCR efficiency than loss of GAPDH rna to target-rna; i.e., ratio of RNA templates (housekeeping gene:low abundance gene) is less important than the ratio of PCR efficiencies (housekeeping gene: low abundance gene).

For more PCR efficienty stuff use the google search in this forum: PCR normalization PCR efficiency or check this --> [Only registered users see links. ]

Lastly, if it is your freezer that is acting up, and you resolve it by changing freezer or whatever; just carry-on. If your samples were in cold storage continously, but the temperature dropped, and samples briefly thawed, just make a note of it and carry on with the experiment and analysis.

Good luck.