The two tall peaks at 2000 and 4000 are the ribosomal RNA, the small one at ~180 is the marker. The RIN is a measure of quality, essentially it's a ratio between the two ribosomal peaks. The second one (4000) should be much taller than the first, otherwise there has been some degradation that occured and the sample is no good. In this sample, the second is indeed taller than the first, but not enough. I actually think it's calculated by area under the curve rather than height, but close enough. Another sign of bad RNA is the ripples in between the two peaks - it should be fairly smooth, when it's rough, that's a sign of degradation.
In all, I was kind of expecting this as the sample thawed briefly before I got it in the buffer. As in the last post, I realized this and planned around it for the next time. Then, mistakenly, I assumed that I should go ahead and start on my samples. I extracted 12 (of 40) and bioanalyzed the first 5:
Each sample here has 2 charts, the first is the totalRNA, the second is the miRNA. There will be no ribosomes in the miRNA, so we have no way of gauging quality with those, but as for the others, they were all pretty awful. No RIN was above 7.3 and most didn't have even an estimate. Clearly there is some degradation although that's not the only thing going on - numbers 7 and 9 both have a weird hump early in the read before establishing the baseline and 6 and 8 have a hump near the end. Neither of these things should happen. This also sucks because they were my important precious samples. I do have back ups for them all so it's not a huge deal, but it's scary as I definitely ruined them and I need to not do this again.
The next step is to bioanalyze the other samples that I've extracted already.
After that I need to figure out what is going wrong:
I will first do another extraction (liver this time - placentas are too valuable) comparing my kit (Omega) with another brand (Qiagen) that we have in the lab and we know works.
- If both come out bad then it's my fault and I need to do better with my technique (Yikes, I really hope it's not this one...)
- If the Qiagen one is bad and not the Omega, then it's an issue with the kit, and we need to get a new kit (or make new buffers).
Another potential issue is that I used standard alcohol when preparing my buffers not realizing that molecular grade ethanol exists. I will be using molecular grade in the future on of course, but in order to avoid confounding variables in the above experiment I will do everything exactly the same for the Omega extraction as I had before. Afterwards, I will do second test will be to use molecular grade ethanol alongside regular ethanol to determine whether the ethanol is the problem.
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