I have to perform qPCR for miRNA isolated from colorectal tumor and adjacent normal tissue samples. Is U6 snRNA a good endogenous control for qPCR?
In my opinion, it is not mandatory to use a non-coding sRNA to control for miRNA expression (though it could be perfectly the case). It should be reminded that miRNAs are transcribed by RNA pol II, much like messenger-RNAs but not for example tRNAs. The processing machinery is quite different for Pol I /II / III-processed transcripts. Besides, to date I do not know about a bona fide housekeeping microRNA.
For these reasons, I use common housekeeping genes, like EF-1alpha, bur you should select the one that in your condition really behaves like a housekeeping, for example looking at Genevestigator or reading related bibliography.
Having said that, I would suggest you to try and compare 3-4 putative housekeeping genes and select the adecuate using Bestkeeper, Normfinder or other software developed specifically to find what housekeeping gene is the most stably expressed in your samples.
Good luck!
Most non-coding RNAs can be used as an endogenous control for miRNA qPCR. so you can take any rRNA, tRNA, snoRNA, snRNA.
I used U6snRNA for miRNA qPCR.
Good luck
In my opinion, it is not mandatory to use a non-coding sRNA to control for miRNA expression (though it could be perfectly the case). It should be reminded that miRNAs are transcribed by RNA pol II, much like messenger-RNAs but not for example tRNAs. The processing machinery is quite different for Pol I /II / III-processed transcripts. Besides, to date I do not know about a bona fide housekeeping microRNA.
For these reasons, I use common housekeeping genes, like EF-1alpha, bur you should select the one that in your condition really behaves like a housekeeping, for example looking at Genevestigator or reading related bibliography.
Having said that, I would suggest you to try and compare 3-4 putative housekeeping genes and select the adecuate using Bestkeeper, Normfinder or other software developed specifically to find what housekeeping gene is the most stably expressed in your samples.
Good luck!
You should experimentally validate whether U6 is a stable gene. In my experience U6 was the least stable gene. Try doing
U6, RNU1A, as well as 3-4 miRNAs that are relatively well expressed in your samples. Have a look on the literature what miRNAs are abundantly expressed in your sample of interest.
Once you have acquired the data, you will need to combine them using a stability analysis software such as GeNORM or the one said above by Carlos.
In my sample heart samples (not related to cancer), I used U6, RNU1A, miRNA-22, miRNA-26, miRNA-103 and miRNA-191.
Hi Bhoomika
Yes. U6 is a good endogenous control for miRNA expression study from tissue or cell line.
And also set of endogenous control also you can strength your study
Please see this link for more information
http://www.exiqon.com/mirna-pcr-controls
Prabu
You can use a combination of snRNAs and snoRNAs in miRNA qPCR assay (if possible) to strengthen your data or validation of true expression. Otherwise U6 snRNA can be used as ref control gene. For your info, in some instances including studies with cancer cell models, small non-coding control RNAs can even vary across cell/tissue types. So it's more acceptable when you have a set of ref genes and take the avg. expression to normalize your specific miRNA level. For more explanation please check attached publication:
Article Altered microRNA Expression Profiles and Regulation of INK4A...
Hi everyone, actually I will first time analyze some microRNAs in tuberculosis patients via qPCR, but I am not sure endogen control miRNAs, I will use synthetic C.elegans miRNA and also some authors used U6RNA just like you said but is this always an endogen control?
This is about my phD thesis and I must teach, Thank you everyone already:)
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