Hello, Can you help with the qPCR analysis, I´m trying to fine-tune...
Hello,
Can you help with the qPCR analysis, I´m trying to fine-tune the primers but I would like to know if in the program for qpcr the melt curve is necessary, because I have already tried several times and I have not managed to get it right. Thanks
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Yes, melt curve analysis is necessary followed by checking the PCR product on the gel to see if you are amplifying your expected PCR fragment. I would suggest to change the divergent primer sets used for circRNA detection. Please design the primers with CircInteractome (https://circinteractome.nia.nih.gov/Divergent_Primers/divergent_primers.html).
Please make sure you are using the PCR cycle conditions mentioned in this manuscript. Using the default RT-qPCR protocol in the real-time PCR machines may create problem since longer time of amplification during PCR can amplify multiple circRNAs containing same exon sequences as your target circRNA.
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