I have a control and one treated sample, and I want to analyze differential expression of selective miRNAs in treated sample as compared to control. But the Reference miRNA/gene is not known.
What will be the best strategy to perform qRT PCR for miRNAs in this case, in minimum possible time with accurate results?
There will be a set of miRNA endogenous genes. Depending on species and experimental conditions, you will get few standard & stable reference genes. That you can search in google scholar & Pubmed. if you provide a little more details, members can also provide you with a list of reference genes.
If the standard procedure has been followed, you will get results. All these things like RNA/miRNA isolation, miRNA-specific cDNA synthesis, proper primers, and a suitable master mix have been used. Then you will get results.
..or you could simlply use as baseline the number of amplifications you consider as the maximum relevant for detecting a probe (e.g. 35) and use as semiquantitative score for your molecule (i): Score (i) = 30 - observed amplifi
cations (i)).
miRNA have often hundreds of targets, but in your case you don't need to know the target to undergo RT-qPCR following the delta delta ct rules with this file.
all the best
fred
Hi Bastir,
From which tissue did you isolate your miRNA? Go to Thermofisher (Taqman assays) https://www.thermofisher.com/order/genome-database/?pearUXVerSuffix=pearUX2&elcanoForm=true#!/microrna/assays/microrna_mature/?keyword=miRNA%20screening&searchMethod=navigators&filters=mirnatype_%5E%22TaqMan%20MicroRNA%20Assays%22$
They have miRNA screening assays in 96 well formal and card. If your miRNA from tissue/serum (human/mouse), go to the screening assay and look for the controls in the layout (there should be exo and endo controls). You can use one of them as your reference.
Good luck
- Badges
- Science topic
- Similar topics
- Biological Science
- Biota
- Eukaryota
- Fungi