Refering a paper we designed primers and did not notice that they used it for RT-PCR . While we tried RT-q PCR with it we did not get any proper amplification with the primers. This made us wonder if there would be any difference in the primers designed for RT PCR and RT-qPCR?
Dear Divya,
The only difference is lies in the length of primers.hope in normal PCR , q PCR primers may work but the size of amplicon is short.
Regards
All the best
Real time Q PCR primer uses Specific sequence primers,
For RT PCR you can have, inespecific hexamers, inespecific primers, or specific primers. make sure what is the type of primer they used in their RT PCR
About your case, secondary structure parameters should be taken into account for RT-QPCR primers, and not so necesary for retro transcriptase pcr, that means, this primers might be too strong to amplify by the means of RT-QPCR , but they do a good work with complementary RNA.
Hi Divya,
The only difference in primers between RT-PCR and qRT-PCR is the difference in size of amplicon it can amplify. You need to check back for what purpose they used that RT-PCR primers. If its used for full length amplification of gene, you cannot use it for qRT-PCR. Even if its amplify, you will not get a proper result in your melting curve analysis. As Carin told, qRT-PCR primers generally design to amplify a short sequence length generally within 200bps. Other than this, there is no difference.
Good Luck
Hi Divya,
I would second every one's opinion. In addition to that check your enzyme fidelity.Check temp. of your enzyme for Rt step.You must denature rt and than only should start cycling reaction, Hope this helps.
Shefali
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