Dear friends,
The picture in the link below shows Melt Curve for the primer I have for the miRNA I am working with. This is the best set up that can accur for all 40 cDNAs. I myself think it is not applicable though the curve is same for all samples. I need your comments. Thank you all.
https://www.researchgate.net/topics.Topics.html?ev=home_xask
https://www.researchgate.net/topics.Topics.html?ev=home_xask
Hi Arash,
In short, this doesn't look good to me for two reasons: A - The peak is very wide suggesting many different products, and B - The peak temperature is quite low. Although this was done using qPCR instrumentation, I would recommend putting the amplification products on gel. Gel electrophoresis is still by far the best way to "see" what is happening in your PCR reaction.
In my lab, it is standard procedure to develop assays using conventional PCR and moving to qPCR only when things seem to be working. Even when the move to qPCR has been made, amplification products are put on gel for a few runs so that the reaction can be further analysed. Only once we know how the reaction behave with this set of primers and probes do we stop using gels.
Sorry, the picture's address is as below:
https://arashazimzadeh.shutterfly.com/
https://arashazimzadeh.shutterfly.com/
Hi Arash,
In short, this doesn't look good to me for two reasons: A - The peak is very wide suggesting many different products, and B - The peak temperature is quite low. Although this was done using qPCR instrumentation, I would recommend putting the amplification products on gel. Gel electrophoresis is still by far the best way to "see" what is happening in your PCR reaction.
In my lab, it is standard procedure to develop assays using conventional PCR and moving to qPCR only when things seem to be working. Even when the move to qPCR has been made, amplification products are put on gel for a few runs so that the reaction can be further analysed. Only once we know how the reaction behave with this set of primers and probes do we stop using gels.
Hi Arash,
Base on this melting curve and Tm for each peak, i would say that your target gene didn't proliferate and these peaks are belong to Primer-dimmer or secondary structures. It could be helpful if you check your target´s GC%, then you can find what will be approximate TM for your target genes.
I agree that the data are not accurate and reliable. As Mark mentioned, run the products on agarose gel, to see the exactly size. I run the qPCr products after each qPCR run, to be sure about non-specific products, even I had test my primers on cenventional PCR.
Melt curve for miRNA?? Do you really mean microRNA or do you mean mRNA (messenger RNA). If mRNA, then the feedback you have from others here is good. I would also consider trying some other proven primer set to check whether there is some problem with the qPCR process or the settings on your instrument.
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