Dear all,
I have problem with my electrophoretic assay of RNA quality. I performed native agarose gel and I obtained no clear result. The marks is very good, but samples look as RNA is degraded.
However, my spectrophotometric assay results in high amounts of RNA and very clear:
RNA conc. 571 ng/ul
260/280 ratio: 2,061
260/230 ratio: 2,078
Tell me, have you ever had such problems? What should I do? Maybe I can skip the electrophoresis and try RT reaction for one sample to check if my RNA is OK or not?
Spectrophotometric analysis only check concentrations for nuclei acids, proteins, phenol, etc, even degraded RNA/DNA can be measured by OD reading. Your numbers are good but they don't demonstrate if your RNA is intact. Since your agaroso gel showed degradation than your RNA won't be good quality grade for RT.
The only assay that can replace the electrophoretic analysis is the Bioanalyzer. The device gives you a RIN (RNA Integrity Number) that indicates the ratio between both rRNA subunits (28S and 18S).
Are you planning on doing only reverse transcription or qPCR?
Spectrophotometric analysis only check concentrations for nuclei acids, proteins, phenol, etc, even degraded RNA/DNA can be measured by OD reading. Your numbers are good but they don't demonstrate if your RNA is intact. Since your agaroso gel showed degradation than your RNA won't be good quality grade for RT.
The only assay that can replace the electrophoretic analysis is the Bioanalyzer. The device gives you a RIN (RNA Integrity Number) that indicates the ratio between both rRNA subunits (28S and 18S).
Are you planning on doing only reverse transcription or qPCR?
In that case you should not go foward. Try extracting new RNA. Be careful with RNAse contamination. Everything must be RNAse free (solutions, tubes, bench top). Are you using Trizol or a RNA extraction kit?
For RNA quality, electrophoretic assays are better as spectrophotometric assay will only tell you the concentration but not the intactness of the RNA. But, combination of both is the best. The sample should qualify in both the assays.
Native agarose gel won't give sharp peaks as compared to denaturing gel analysis. Secondary structures of the RNA will give you different migration pattern even on repeat analysis. Did you heat your sample before loading?
Best alternative would be to run using Bioanalyzer or TapeStation as they will give you an objective quality score indicating the intactness. If that is not available, try running the samples in denaturing MOPS /formaldehyde gel, also ensure to add the dye both in gel and running buffer for better visualization.
How did you make your native agarose gel? What percentage gel? I usually make a 1.5% of TAE agarose gel to see clearly rna bands after spectrophotometer assay.
To test integrity you must either perform agarose gel electrophoresis and see clear rRNA bands or use bioanalyzer which will tell you the integrity of your RNA. Spectrophotometer simply tells you the concentration but cannot determine the integrity of your RNA, which if degraded would produce a smear on the agarose gel. I would not proceed with the sample that does not show up as two clear rRNA bands on the agarose gel. Good luck!
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