Is it supposed to be added along with my miRNA primer in the same pool of RNA? Lets suppose I am checking the expression of mirX microRNA and I have 2 conditions- treated and non-treated. So per reaction, i will have to add U6 control primer along with my Taqman probe for each condition? Also, do I have to run a reaction with just U6 primer?
No, you have to perform RT reaction separately to prepare cDNA by U6 & your target miRNA(X) specific primers for each experimental group( Control/ treated) and then perform Real time PCR reaction. For example, if you want to check the expression of miR-21 in one control and one treated group then you have to set up Four cDNA reactions, two by U6 specific RT primer for both control & treated groups and two by miR-21 specific RT primers or both control & treated groups. and then perform Real-time PCR reaction for all four cDNAs by U6 & miR-21 specific TaqMan Probes respectively.
For cDNA preparation, you can multiplex with many primers, like to run TaqMan low density(TLDA) array we prepare cDNA of 384 miRNAs in the same reaction but you need to standardized your program for multiplexing, For individual miRNA assays preparing cDNA separately is a more standard procedure. And for Real-time PCR, yes TaqMan probe is sequence specific but for detection of multiple targets in the same reaction you need each probe labeled with different fluorophores to prevent overlap of signals.
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