Recently I am engaged with the transcriptome analysis for differential expression of genes of a WT strain vs. its mutants (is a fungus). Mutant strains grow poorly; it is difficult to obtain good quantities of RNA, and even when the peaks are clear in RIN analysis, they are below 7 (between 6 and 7), In order to obtain even results I have obtained RNA from the WT strain having RIN between 6 and 7. I have read about RIN sometimes could be used below RIN = (7 or higher) standards, for example from paraffin embedded samples, however I need the results would be publishable, so I should be very grateful if you could recommend me additional experimental analysis to demonstrate the RNA is useful. Thank you very much.
In case of fungi It's difficult to get good quality RNA but in my experiment of Ground Nut Gene expression analysis against A flavus Fugus.
I used Gen-Next MN kit and tried trizol method which gives quality RNA .
I would believe that RNA samples with a RIN number between 6 and 7 are of reasonable quality, unless certain RNA species are preferentially degraded in one strain but not in another strain during your RNA preparations. So, what is important is the difference between your WT strain and the mutants. Having said above, I think you can perform a Northern blot analysis, probing for a number of RNA species just to see if they show a clear sharp band after gel electrophoresis. If so, you are further assured that your samples are of good quality.
For all gene expression analysis the use of multiple reference genes is advised anyway. So you could increase the number of reference genes (aka house keeping genes) and perform qPCR. As long as these genes show a similar expression in you test samples the expression of your genes of interest or entire GW transcriptome is publishable. Certainly if you describe all steps in detail.
Bas Brinkhof , Hua Xiao , Jayale Ashwin Sanjayrao :
Thank you very much for your advices, I'm sure they will work very well.
Every point was remarkable.
Nice work, and best regards.
Patricia
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