Hello, Can you help with the qPCR analysis, I´m trying to fine-tune...

protocol Protocol: Detection and Analysis of Circular RNAs by RT-PCR
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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Amaresh Chandra Panda Author Answered Apr 1, 2019

Institute of Life Sciences Bhubaneswar

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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