I'm using primers and reagents from Qiagen to detect some microRNAs of my interest. I saw some publication using the same thing, but none of them showed gel images (run PCR product on high resolution agrose gel). Is there anyone who has done this or a melting-curve test? I ran a gel but got double bands even for housekeeping gene RNU6B. Is this normal?
Hi Yiwei
I am doing microRNA detection, every experiment I do melt curve test.
RNU6B and my microRNA melt curve shows at single temperature only.
RNU6B cut value is 18- 21.
But I didn't ran gel.
My suggestion while doing Qpcr include ( positive and negative control).
Pp
I think running a gel to see a single band with predicted size would be more convincing than a melting curve.
Hello
Agree with Prabhu Paramasivam , the papers & link here may help you in your question
Good Luck
http://www.filetypepdf.com/qi/qiagen-microrna-protocol-pdf.html
Hi Eve
I've used that particular system, and whereas the miRNA reverse transcriptase reagent works nicely, I had much better PCR efficiencies using non-Qiagen SYBR reagents (e.g. Quanta and KAPA). In order to validate the PCR products (by Sanger sequencing) I cloned the PCR products into the pGEM vector. This allowed me to read the entire PCR product instead of getting a read from ~20bp downstream the sequencing primer.
PS: If you decide to run a gel, keep in mind that the relative small bands resulting from miRNA RT-PCR is better resolved on a PAGE gel than even a high percentage agarose gel.
Thanks Kristian! I also tried another Sybr Green Mixture (although also from QIAGEN) and got much higher efficiency! However the difference between treatment and control groups became smaller. I also though about cloning it into a plasmid vector and sequence it! Good idea!
We found minor difference in Ct cycle number (0.5~1.5 cycle) between control and treatment groups for the miRNA of our interest, but this increase, though small, is consistent and reproducible. In addition, we always observed that the maximal Cyber Green signal amplified the in treated group is much higher than that in control cells. Does this mean something for our treatment intervention? I mean minor cycle difference with significant difference in the maximal Cyber Green signal.
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