Hi Jonna,

Looking at your Tapestation report is obvious that your sample is completely degraded. I don't know the methods you use for measuring 260/280 and 260/230 ratios but these measures do not represent what RIN tells you. I recommend you use Epicentre kits for RNA isolation. I have very good results for bacteria and eukaryote cells. Important things to consider during extraction: RNAse-free tubes (Eppendorf or Ambion), sample mixing steps by tube inversion instead vortexing, use RNase-free DNase I enzyme and short time of incubation. I also reccomend you to use RNase-free water (Ambion) for final RNA suspension and avoid repeated freeze-thaw cycles.

Good luck!

Hi Jonna, RNA quality assessment has two aspects: purity and integrity. You measure RNA purity through 260/280 ratios and RNA integrity through Tapestation (28S/18S rRNA ratios). So you can have very pure RNA (260/280 from 1.8-2.0) but at the same time the sample can be completely degraded. In your case your RNA sample is very pure, but it's completely degraded, likely due to RNAses as Alfonso mentioned. I don't use kits to isolate RNA, but from what I've heard they're quite good (better than Trizol which I use). Just use RNAse free tubes and filter tips, keep your RNA on ice and analyze/ process it quickly. If you're making cDNA try to do it on the same day you isolate your RNA and have its 260/280 and RIN scores. Don't forget to elute the RNA in RNAse-free water or DEPC treated ddH2O. RNAses are one of the most abundant and robust enzymes out there and they even re-nature after being autoclaved!

Thank you for your answers! I was not sure if I had contamination or if the curves were due to degradation. We have tried to keep the RNA extraction lab free from DNA/ RNAses, but the work space is not optimal. I have used throughout RNAse free eppendorf tubes and the resuspension with RNA-free water. However, there are a few steps in my CTAB protocol that could be the cause of degradation. Perhaps you could help me find those.

Firstly, CTAB buffer is preheated in 65 C. The liquid nitrogen grinded sample are then to be added, and incubated in 65 C for 10 min. I am not sure if heating up the sample is a good idea. What do you think?

Secondly, adding chloroform-isoamyl to the buffer and shake vigourisly. In the protocol, it is supposed to form emulsion. But as mentioned shaking the sample would fragment the RNA, even DNA to some extent. 

My most consern is the heatening, should I keep everything more to room  temperature and centrifuge (4C)? Also what should the maximum speed for the centrifuge be?

Best regards,

Jonna

I've never done extractions with CTAB, but I read a protocol from this article and they just use room temperature CTAB: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2813910/. I would avoid heating the sample too much. In my protocol for Trizol I freeze the tissue samples in Trizol and thaw it later when I want to do the extraction on ice and grind the tissue in the Trizol (RNAses are inactivated in Trizol, and CTAB with beta-mercaptoethanol). In that article they get good RIN scores with their CTAB protocol. 

Thank you so much! I will avoid the heating and see if that is the main problem.

Hi to all,

how can assign the peak 18s or 28s on the elettropherogram? On the basis of how assign the peak, the quality (RIN) change.