Hey guys,
I've been struggling with quantifying miRNA with real-time PCR and the problem now i have is that i couldn't get consistent results. I tried to repeat the experiments literally two days in a row, starting from RNA extraction all the way to realtime pcr but the results are so messed up and totally different. I am using mirVana RNA extraction kit from Invitrogen and followed by QuantiMir kit from SBI. I would appreciate any suggestion. Thank you guys!
Hi,
I also use miRVana kit, but to have the reproducible results I think it is better to try TaqMan microRNA Assay which is also from Invitrogen. TaqMan-based kits give you better results as compared to SYBR-based ones, especially when the expression level of your miRNA(s) of interest is low.
hi xingchen
i have also used same quantimirkit
but i used cell to ct ,plus quantimir
like ur experience we also had similar problem as i isolated mir from mirnasey kit and used by quantimir gave inconsistent result
we decided to use cell to ct quantimir way ,it sorted out problem we got consistent result
there were recent report about trizol or similar based isolation methods ( here mirvana in ur case ) in which the mirna isolation is biased as per gc content of mirna ,so we decided to use cell to ct kit
Hi Xingchen.
In the lab, we use gold standard trizol (quiazol) extraction followed by DNase 1 treatment, and after for the RT we use the kit from Quiagen miScript II RT kit, very easy steps to have reproductible consistend result, if you need more information or help just ask
Hi Xingchen, what is the problem, how about not consistent results?
expression level of miRNA o do you think it depends on the extraction method? I also used in the past mirVana extraction kit and it worked perfect...
Hi,
I also use miRVana kit, but to have the reproducible results I think it is better to try TaqMan microRNA Assay which is also from Invitrogen. TaqMan-based kits give you better results as compared to SYBR-based ones, especially when the expression level of your miRNA(s) of interest is low.
Hi,
I had used this quantimir kit and now moved to Mir-X kit from clonetech. I would suggest you to use TRIzol prepared RNA and Mir-X™ microRNA First Strand Synthesis Kit (#638315, Clontech) for RT. This is a simple kit with one-step reaction which reduces chances of variation between experiments.
I also use Trizol, I have found it to be the best for miRNAs. I have had great results with the polyA miRNA kit from Gencopoeia, much cheaper than Taqman or anything from Qiagen.
Hey Xingchen,
Could be a number of issues not related to extraction or PCR method. Are you extracting from in vitro or in vivo cells? Are you storing your RNA correctly, especially avoiding freeze/thaw cycles? How are you measuring your RNA yield? Are you consistent with your RNA input into the reverse transcription step? What are you using as a reference gene for RTqPCR? Its important to be consistent with all the above steps, but also essential to have the right reference gene (2 is always better than 1).
Daniel
we extract rna with trizol (ambion), miRNA cDNA prep using stemloop primers and RevertAid Reverse Transcriptase followed by SYBR Green assay using KAPA SYBR Universal kit and fordard primer + universal reverse primer. Results are consistent when U6 SNRNA taken as reference gene.
Thank you for all the replies! Yeah i know that Taqman miRNA assay from Invitrogen is legit but that based on the fact that you know which specific miRNA you want to study. Actually i am doing a 7miRNAs screening in which case I knockdown a gene and look at which miRNAs' level changes. That's why i choose the SYBR method just to save some money.
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