I'm guessing you followed the full isolation procedure for both Trizol (including an alcohol precipitation step) and then the RNaqueous kit (including a lysis buffer)? I think you could streamline this quite a bit by using column kits (rather than a syringe kit) which are designed for smaller quantities of starting material. Since your RNA is complexed with an RBP, I definitely agree with using TRIzol as a first step to denature the proteins and release the RNA. From there, I would suggest trying the Zymo Direct-zol RNA microprep kit, which would simplify your extraction a lot because you can just put your sample dissolved in TRIzol directly into the column (with no need to add chloroform or perform phase separation or RNA precipitation). This procedure is very easy and I think it would give you much better yield and purity compared to trying to precipitate the RNA and then clean it up using a kit which is designed for sample lysis rather than RNA cleanup. I really like the zymo kits because they are very easy to use and the column design is excellent, but many other manufacturers make an equivalent kit which is designed for direct input of RNA in TRIzol or similar reagent directly into a column. Any of them would likely work for you. I strongly recommend including an extra centrifugation step (2-3 minutes at full speed) between the final wash and the RNA elution to remove residual ethanol from the column, it will tidy up your ratios even more.

I also think you could rescue the samples you already have rather than making new ones. It looks to me as though you have quite a lot of RNA actually :) Assuming you have at least a few microlitres at ~150ng/uL, you have plenty of RNA for library preparation without the need to use a low input library kit, it just needs further purification. I would use Zymo clean and concentrator 5, but again most manufacturers will have an equivalent.

Whether you decide to cleanup your samples or make new ones (or both), if you possibly can, I suggest checking your RNA concentration with a fluorometric method (eg Qubit) and/or checking your mRNA integrity on a BioAnalyzer before you use them for preparing NGS libraries. Nanodrop is not very accurate. In my experience, time spent on quality sample prep for NGS is repaid five-fold when it comes time to analyse the data. Best of luck! :)

Hi Laura,

Thank you so much for your feedback. I will definitely try the Zymo Direct-zol RNA microprep kit. But to let you know, I haven't done the entire protocol of TRIzol. I only isolated my RNA from the bead+protein complex and then directly used the RNaqueous kit (which contains column) to purify my RNA but the sample appeared contaminated. And from what I understood based on your recommendations, that this procedure should work fine with Zymo Direct kit.

Thank you once again :D