Hi, I have a problem to get a good RIN value from my RNA isolation for mirna research, I did it with qiagen mirneasymini kit, and trizol method (with different trizols for example I used qiazol for one time in mirneasy kit and once trizol), I used pelet from a college and she store it in PBS at -80. (RIN is nearly 2.5 from mirneasy kit or trizol method; the same kind of pelets give good RINS near to 9 when RNeasy kit is applied)
Having a good RIN value to study miRNAs is relatively important... As Prabu up here says, it's a relation between 18 and 28s peaks... If your sample is not from cellular RNA, don't expect to have a good RIN, mine is usually below 1...
Besides, miRNAs are extremely stable, like, ON at RT stable, so if that's the only thing you want to study, I'd say go ahead and then try to get a subset of quality when you validate them... maybe not the best approach but not the worst. Also, RNUs are much less stable so take that into account when choosing your housekeeping genes...
RIN is just indirect method to check the quality of you miRNA because the value is shown the ratio between major RNAs. So I think the best way to control miRNA is to do RT-qPCR by target's copies check. Good luck!
RIN values below 7 confirms the RNA as degraded. Please reduce the ways of RNase contamination by taking all the precautions for RNA isolation. Degraded RNA samples may interfere the sequencing procedures and may be getting false positive results.
Did you isolate pure miRNA with miRNeasy kit or total RNA including miRNA?
Do you use glycogen as a coprecipitant with your Trizol isolation?
Storing RNA in PBS would not be my advice. Why PBS?
I want to add to some answers: The RIN of course depends on the 28S/18S ratio, but is not the same. The RIN algorithm takes the whole electropherogram into the calculation. Besides this, the Bioanalyzer does not run under denaturing conditions so it does not accurately give 28S/18S ratio's. I have had samples with RIN around 8 and 28S/18S ratio of
My experience is that RIN has no bearing on miRNA qPCR results. Se an earlier discussion: https://www.researchgate.net/post/Is_RIN_important_when_analysing_microRNA
In my clinical samples I had RIN values between 1 and 9. There was no correlation between miRNA expression and RIN for 32 different miRNAs. RIN was developed as a marker of degradation of mRNA, which are far longer than microRNA. Se the attached original article by Schroeder
That is why I asked if it concerns pure miRNA or Total RNA. With pure miRNA it is not possible to calculate a RIN.
Again, it depends on source of total RNA... exosomal, mitochondrial, circulating... all have different profiles and their stability cannot be judged based on a RIN value.
I don't work with RNA from these sources Jennifer. I wonder what kind of quality check you can do then?
Depending on what's the experiment that you are interested on doing you can do one or another test...
If you are interested on microRNAs, they are extremely stable so it's unlikely that you will have them degraded, so the main problem is then to actually normalize the amount of RNAs / test, and you can do that by checking the levels of one miR that you know is stable among conditions.
If you are interested on mRNA, then you can by PCR detect several RNUs and housekeeping genes. Though RNUs are less stable than miRs they are more stable than mRNAs... so if you see that the relationship is not stable in all your samples, bad news... You can also make a relationship between miRs/RNUs/housekeeping...
I haven't understood from where you isolate RNA from? cultured cells or tissue?
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA