We're aiming to perform RNA-Seq using RNA from FFPE samples. We've been using the RNeasy FFPE kit (from Qiagen), which is often recommended, and we've also tried the miRNeasy FFPE kit to try for microRNA-Seq.
We analyzed the RNA using TapeStation to acess RIN/DV200 values and did not find significant differences. However, the majority of fragments are below 200 nt and we are afraid the quality will not be enough for this technique.
Has anyone had sucessful results? Did you optimize the extraction protocol or did you follow the manufacturer's recommendations?
Thank you.
It is unclear which kit you are using for RNAseq, I assume QIAGEN's kit but I could be wrong. I would consult the kit's handbook to see what the requirements are for the library. Usually, it is not a problem to have smaller fragments for RNA especially for counting applications where 75x1 read protocols can be seen. Take into consideration the protocol's read length requirements and what would happen if you couldn't meet it. Usually short transcripts are mappable, but if you're looking for RNA fusion transcripts you could stand to gain from longer paired-end reads (150x2, 230x70). It seems that you are doing microRNAs, which microRNA type you intend to pick up with the kit? The pre-microRNAs or mature microRNAs? The former is longer and the latter is a shorter transcript hence their mappability could differ depending on your fragment lengths.
As per your comment, "did you follow the manufacturer's recommendations" - always, always follow the manufacturer's protocol. They (should) come optimized and you shouldn't have to make tweaks which can be costly for time and reagents. I would also encourage you to engage the local field application specialist for the kit that you intend to use, they should have some experience or have colleagues that can comment on your specific situtation.
Can Kiessling Thank you very much for your answer. I mentioned the manufacturer's recommendations because they are a bit confusing. In the handbook they mention no agitation during the 56 ºC and 80 ºC incubation periods, not even in the troubleshooting guide. However, in the quick start protocol provided in the kit, that step mentions agitation or vortex every 3-5 minutes. I'm not sure whether I should agitate it or not because I've read other kits mention agitation could promote RNA shearing or degradation.
You should stick to what your kit says, mixing and matching steps from other kits is not a good idea and can (and will eventually) lead to pain. Manufacturer should know under what conditions their kit will provide results - they are beholden to it so long as you execute their directions to 100%. Remember, kits can have different way of doing the same thing with their own proprietary chemistry. If you stray from the protocol, they will not be able assist in troubleshooting because you did not apply the protocol 100%. I worked in clinical testing as well as QIAGEN, I have seen this happen and you wouldn't to be in that situation as a customer.
Can Kiessling Thank you, but I actually mean the protocols from the same kit are conflicting - between the handbook and the quick start protocol provided.
In that case, you should definitely follow that up the manufacturer (or if someone had found how to get your kit to work - either using the quick start or the long protocol). Only they can give clarity on which protocol(s) would work. You should transfer the liability to them.
- Badges
- Science method