I am trying to design a primer for mRNA targeting a gene having size of approx. 6kb. So, I am choosing its exons to make the primer but they show hairpins and mismatches along with a small primer product (~120 bp) which can probably form non-specific. Can I add intronic region as well but it gives 2kb product size in insilico-PCR. What should I do?
Hi Fatima
stay on the mRNA sequence for designing primers for qRT-PCR, since if you take some primers in introns, you'll amplify premature RNA or genomic. it depends on your protocole for RNA extraction, but typically, mRNA do not contain intronic sequences.
take primers at the most 3' of the gene to get fixed with RNA degradation. a size of 100-150Bp amplicon is the best in this case, with almost one primer across 2 exons splice. take th best from the worst, no choice, adding DMSO at 5% with higher Tm should work.
all the best
fred
No, you do not include the introns for qPCR primers.
Ideally, one of your primers would "span" an exon-exon junction so that it's not possible to amplify a product from the cDNA. Any amplification is from gDNA contamination.
Next best is to "flank" an intron with the forward and reverse primer so that the cDNA & gDNA give different size products (can tell if you have contamination from the gDNA).
Last choice is primers that are in a single exon. As long as your DNase treatment is complete & all controls work, then those primers will work.
Finally, you WANT to have a very small (~120 basepairs) PCR product for qPCR. Products that are too large don't amplify efficiently.
Talk with a colleague before you do any more designing to make sure you fully understand your experiment.
Good luck!
No, it is not recommended to add intronic part of the gene with exonic region to design primers for RT-qPCR. Primers that anneal to introns will not amplify the target gene.
It is best to design primers that anneal to the exonic regions of the gene. As a result, primers that anneal to exons will amplify the target gene. Makes it easier to design primers that will amplify the target gene.
When designing primers for RT-qPCR,
choose primers that are at least 18-20 nucleotides in length. Make sure that the primers have a melting temperature (Tm) of 55-65°C.
Design the primers so that they anneal to regions of the gene that are relatively conserved between different individuals and use a primer design tool.
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