In our diagnostic lab, we extract pure viral RNA (Qiagen viral RNA extraction kit).
Therefore, no 28S and 18S rRNA can be traced in agarose gel for total RNA integrity assesment. However, very small bands appear on gel (as seen in picture), are those 5S rRNA or other small RNA moleculea? If so, may they be correlated to total RNA integrity?
Is there any other way to asses the pure viral RNA integrity (besides Bioanalyzer)?
Thanks
Hello Ido From
Other than Agarose gel and Bioanalyzer, you can try real-time qualitative PCR (RT-qPCR) and RT-PCR. Delayed Cq values (quantification cycle value) could suggest poor integrity. I came across the following article. Hope this would help.
https://academic.oup.com/jxb/article/63/17/6069/564165
Best wishes!
You can use primers that amplify products of different lengths. In low-integrity samples you then observe an increased shift between Ct values of shorter and of longer products. I used this to get a hold of RNA integrity of laser-microdissected samples where the amount of RNA was not sufficient to see peaks in electropherograms ("Bioanalyzer") and not to mention bands in agarose gels. As I used oligo-dT priming in the RT, I amplified equally sized products that were located at different distances from the poly-A tail. If you use randompriming, it should work also with the amplification of products of different lengths.
You will need a standard of known good quality to compare against. If nothing else works, you can make a synthetic standard by mixing purified PCR products (in equimolar ratios). But take care of cross-contaminations (prepare/purify the standard in a different lab, ideally in a differn building!).
@Jochen Wilhelm your answer is very insightful and enlightning. In my case- I study avian gamma Corona viruses (IBV) which have highly variable genomes, so designing more universal primers (other then the one published set we and most of the diagnostic laboratories in the world use) is not an easy task.
Thanks a lot for your answer, I will be happy to further hear any other idea that mught help in this challenge.
Hi Ido
Using QIAamp viral RNA mini kit, using carrier RNA, you would get RNA fragments greater than 200 nucleotides, therefore, it may not be 5s rRNA which are less than 200 nucleotides. I am not sure about the size of RNA ladder that you have there. You may try spinning down the cells and other possible contaminants in sample at 1500xg for 10 min at 4 degree C and use the supernatant for viral RNA extraction.
You may check the integrity of viral RNA using Qubit Fluorometer (the assay is not very expensive)- don't worry about the RIN value as there is no 18s and 28s rRNAs, rather look at the viral RNA peaks and size of the fragments in the image it provides. This will definitely give a clue on the quality of your RNA prep. I hope it helps.
Thanks @Ravendra P. Chauhan.
Yes the ladder in that gel was not good, but on other gels we see that the length of this fragment is around 200 bp. The presence of this band is also seems to be correlated with succsessful amplification of the target viral gene (compared to samplea without it). I really appreciate your answer, I didnt know the minimal size of RNA fragments that can be eluted with the kit. We also consider purchasing a Qubit.
Thank,
Ido
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