Hi Everyone
I have tried using U6 and 5.8sRNA as endogenous control. Although the expression of both these remain constant in different conditions, the Ct values for U6 are quite high (30 or so) and that for 5.8s is low (15-16). I tried increasing the concentration for U6 and decreased for 5.8s but still the ct values are almost same.
Is it advisable to use any housekeeping gene as endogenous?
Any help would be appreciated.
Thanks
Hi Swati
The answer is that you can use those genes depending on your particular study. You can use U6 and 5.8S RNAs with no problems. In your case I will bet for the one that has the lowest Cts, however you must carefully look around your samples to see if this RNA has a relatively constant expression. You can also try some SNORDs and tRNAs (lysine and arginine tRNAs), if everything fails.
Good luck. All the best
Paco
Hi again Swati
I forgot to say that for miRNA quantification by qPCR you must try to find some small RNA as normalizer. For this reason small RNAs as U6, tRNAs, etc are more adequate than GAPDH for example.
Best. Paco
You may also use Actin and 18S as reference genes to your analysis.
Regards.
Hi Swati
You should use small endogenous control genes including snRNAs, snoRNAs and miRNAs for normalize miRNAs qPCR. I know some miRNAs such as miR-16 , are suitable endogenous control genes for some of human cell lines and tissues samples, you can search some candidates for plant samples.
Good luck, best regards
Thanks everyone for replying promptly to my query. I am going to try again 5.8s albeit by changing the dilutions.. Hope it works.
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