So - my lab did a bunch of RNA isolations from tissue culture cells, for purposes of RNA-seq, and submitted them to a core facility. We got back a notification that the RINs were too low, and we should submit better quality RNAs. Now, before I sent them off I ran them on a gel, and to my naive eyes, because I don't run RNA gels very often, they looked okay - I could see rRNA bands, and there was no apparent degradation, i.e. smeary bands at the bottom of the lane.

BUT: since the last time we did RNA-seq, which was around the beginning of the pandemic, and now, we have changed our RNA isolation procedure. Previously we used QIAGEN RNeasy, with On-Column DNase, but I find that protocol very tedious and it's ridiculously expensive, so I moved to a method that I'd used in the dark ages (early 2000s), of (1) Trizol lysis -> (2) isopropanol precip -> (3) DNase digestion -> (4) ethanol precipitation. This gave much better yields than RNeasy, and worked great for RT-qPCR. Now, from reading online, I knew that RNA-seq requires higher purity than you get from a precipitation-based Trizol protocol, so for these samples I went from DNase digestion to a column purification kit (EZNA Total RNA Kit I, which multiple papers used for this purpose). The elutions from these is what I had run on the gel, and what I sent to the core facility.

Now, having been told that our RNA sucked, I redid some RNA isolations, doing the Trizol/DNase/EZNA method side-by-side with RNeasy/On-Column DNase, in parallel on the same cells. The image of the gel I ran is linked below, lanes as follows:

1. DNA ladder 2. RNA ladder 3. "positive control" total RNA (lol - don't buy Fisher 43-072-81 for this purpose) 4. one of my old RNAs samples, made with Trizol/DNase/EZNA, which gave a RIN score of 4.0 5-6. newly-prepped RNA samples, using Trizol/DNase/EZNA method 7-8. newly-prepped RNA samples, using RNeasy

Looking at the gel, it's very likely that the RNeasy samples (lanes 7-8) would give a higher RIN score, because a big part of the RIN score is the 28S:18S ratio, and there's more 28S rRNA in those lanes. But when I look at lanes 4-6 (Trizol method, old samples vs new), I don't see a clear difference in overall quality from 7-8 - there's no increase in low m.w. RNA, and the overall smeariness looks comparable. Instead, it looks like there was preferential recovery of RNA below the size of the 28S rRNA, which would thus produce a lower RIN score.

So, tl;dr version: is there any reason to think that the Trizol method might favor lower-size RNAs, which could produce an artifactually low RIN score? In other words, could the difference between my samples be explained by size-biased recovery of RNA, rather than degradation? Has anyone here ever seen anything like this? And looking at the gel, would you say that we should re-isolate all our samples using RNeasy (which will be a pain, as there were 24 samples total from 4 different cell lines), or would you expect that RNA-seq should still be interpretable despite the low RINs? (I know, I know, we are already thawing the cells to redo it all because why spend the money when you're in doubt, but I've never been a big fan of the Bioanalyzer methodology and I hate QIAGEN.)