microRNA exiqon individual assays - (Mar/27/2012 )

Hey there everybody!

I am writing because i have some doubts on an experience of my own. I am performing qRT-PCR for exiqon individual assays , and i want to get an absolute quantification of microRNAs in my samples. My doubt is: what RNA sample do i use to perform the standard curve? i am currently using reference rna from prostate tissue (Ambion) because my work is related to prostate tissues, but some of the miR's i am testing are not amplifying neither in samples, which would be plausible if they are not expressed in tumors, nor in the standards. Any tip?

Thank you,

Pissitu

As long as the target microRNA is expressed at a basal level, that makes a good sample for standard curve. If the specific microRNa that you are working with is known to be a low-abundance microRNA, then you need to increase the starting material. For Exiqon, you need at least 100 copies of your microRNA to be able to detect it with SYBR green.