Hello,
I'm isolating total RNAs, as well as fractions (large and small) using mirVana (Ambion) in order to obtain circRNAs (via RNaseR digestion). I wanted to validate the samples with RT-inversePCR method before further enrichment (to find out if circular forms are already there). I performed RT with random-hexamers and 2,5 ug of total RNA, followed by an inverse PCR with already validated, specific primers. Did not obtain satisfactory results. I previously used RNeasy (Quagen) for this purpose and RT-PCR confirmed the presence of circRNA each time. Switched to mirVana due to highly increased yield. I previously ran RT-PCR on mirVana-extracted material and it went correctly. The only difference between the subsequent manipulation was storage: in first case, the sample was processed (RT was performed) immediately after RNA extraction, in second case the sample was stored in -80 C for three weeks. RNA samples were quality-controlled on each step (Qubit, Implen, denatouring electrophoresis). Each time sample was fine. The bands were sharp, intense, on appropriate height. During the manipulations I have taken all precautions required for working with RNA and ended up with nothing.
Does anyone have similar experience? Can anyone solve the problem?
present of RNase may cause degredation of RNA so RNase inhibitors shoulde be added to plasma sample
I agréé with Dr Muthana, you should add Rnasin to your plasma samples to avoid Rna dégradation. Good luck.
Hi, thank you very much for the answers. As I already mentioned, all required precautions were taken. The Ribolock (Thermo) was added. Sterile plasticware (RNase free certified) was used, as well as RNase free water (GE healthcare). I worked in a chamber with laminar flow (UV-irradiated before the extraction and RT-PCR). The RNA bands stained with midori green were fine after electrophoresis. There was no smear, Qubit also indicated consistent results with implen. The sample was not degraded. It looked perfectly fine. Which is why I took it to the further steps.
We do typical Trizol extraction and RNaseR assay as a validation of circRNA candidates. we never had such such problems, only caution to be taken is with RNaseR. if it is unopened, using for the first time you would definitely see very harsh activity of RNaseR and it effects circRNAs in some extension, where as if you are using RnaseR from already used (opened) RNaseR enzyme vial it is not such active to degrade circRNAs. so i guess it is not a problem from your sample storage and preparation method.
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