We are interested to analyze the expression of a test gene by Real Time-PCR. Primers and PCR conditions were the same as in a previous paper with the same organism but in our hands the product is not obtained. Primers of housekeeping genes work well. What do you recommend to overcome this?
Regards
Raúl
First check that the primer sequences are correct. There are many instances of authors publishing the wrong primer sequences and even one mismatched base near the 3' end of a primer will stop amplification. Then check that your organism does not have a common snp under the 3' end of one of your primers and that your organism does express this gene
I have been through this too, and sometimes they just don't work, just because a primer is published doesn't mean it will work. But before you discard them you can try some things. I agree with Paul, first check in PrimerBlast if the primer sequences are specific to your target, if they do, then try different melting temperatures, you said the PCR conditions were the same and even if the sequences are the same the Tm can vary between laboratories. Also try to standardize them in a sample where the gene is highly expressed, sometimes you can't see it just because it isn't present in the sample. Hope this can help, if doesn't go for Oscar's advice.
I agree with Daniel. You check the primer target seq, specifically the binding site, and check the BLAST E value. Your Primer sequences might have another target. Then, try to standardize your annealing with gradient PCR.
- Badges
- Science topic
- Similar topics
- Methodology
- Research Methodology
- Research Papers