Hello, I would like to know your opinion about it: apart from checking the amplification curve, what parameters are considered to validate the primers in a singleplex type real-time RT-PCR?
I designed three primer pairs against dengue virus serotypes 1,2 and 3 and plan to adapt it to a CDC real-time RT-PCR assay.
I am new in this type of studies for validation.
Please, if you know, you can send an email: [email protected]
Thank you very much.
Hello Joel,
You can do a melt curve analysis and make sure the primers are specifically amplifying your gene of interest as well as has a single peak. Additionally, you can run an agarose gel for the products and confirm the amplification.
The link below could help you:
Article Guidelines for validation of qualitative real-time PCR methods
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