I am running qRT-PCR for quantification of gene expression in Cyanobacteria. The designed primer is showing single melt curve at Wild type's sample but it is showing multiple curves in sample from mutant. The primer were checked for spicificity by doing simple PCR and showed single band indicating good priming capacity. Before making cDNA, the RNA sample were treated with DNase1 to remove DNA. The picture is attached below. Some suggestions are higly appreciated.
It's pretty common for qPCRs (even with very tight specificity) to present primer dimer formation...eventually. Given primer dimers are usually short, they present as melt curve peaks of much lower melting temp (which is what I suspect you're seeing here).
It's not ideal, but it also doesn't necessarily have to be a problem, as if primer dimer amplification only occurs relatively late (i.e. in cycles after your measured Ct) it will not affect your measured Ct value in any real sense. The melt curve shows you the melting temps of ALL double stranded DNA species in your well at the completion of the reaction, but it doesn't tell you when they actually appeared.
Also, the level of primer dimer formation in your first curve is clearly pretty low, suggesting either a late appearance of primer dimers, or a very low efficiency of primer dimer amplification. The sharp peak at ca 79 degrees (most likely) corresponds to your actual qPCR product, so I would be tempted to use this data.
In the second melt curve, what you're looking at is essentially what happens when there is no template to amplify. Your first samples clearly show that your primers CAN amplify your template (with slight primer dimer formation), so your second shows you what your primers would be expected to do in the absence of any template: non-stop primer dimer formation. In the absence of anything specific, everything nonspecific gets much freer rein.
Long story short: your gene is expressed in your first sample, but is either not expressed, or is expressed at a greatly reduced level in your second sample.
With respect to your conventional PCR:
So. Easiest fix is to simply order a different set of primers. I usually order two or three different primer pairs for each gene of interest, because primers are cheap and sometimes a given pair...simply doesn't work as well as it should.
Alternatively, you could play with your cycling conditions to try and enhance the specificity (higher annealing temperature, shorter extension time, etc). Primer dimers can often be avoided by a simple 1 degree increase in annealing temperature.
there is a possibility that mutant strain would expresses a similar sequence so your primer misspriming them as well as your target gene. but comparing your two set of melting curve it is clear that extra pics have lower Tm than your main pic and this is sign for primer dimer formation. if your mutant strain have lower expression rate, your primer could not find enough template to amplify, thus primer dimer would form. run your mutant PCR on gel and find out products size to be sure about dimers.
It's pretty common for qPCRs (even with very tight specificity) to present primer dimer formation...eventually. Given primer dimers are usually short, they present as melt curve peaks of much lower melting temp (which is what I suspect you're seeing here).
It's not ideal, but it also doesn't necessarily have to be a problem, as if primer dimer amplification only occurs relatively late (i.e. in cycles after your measured Ct) it will not affect your measured Ct value in any real sense. The melt curve shows you the melting temps of ALL double stranded DNA species in your well at the completion of the reaction, but it doesn't tell you when they actually appeared.
Also, the level of primer dimer formation in your first curve is clearly pretty low, suggesting either a late appearance of primer dimers, or a very low efficiency of primer dimer amplification. The sharp peak at ca 79 degrees (most likely) corresponds to your actual qPCR product, so I would be tempted to use this data.
In the second melt curve, what you're looking at is essentially what happens when there is no template to amplify. Your first samples clearly show that your primers CAN amplify your template (with slight primer dimer formation), so your second shows you what your primers would be expected to do in the absence of any template: non-stop primer dimer formation. In the absence of anything specific, everything nonspecific gets much freer rein.
Long story short: your gene is expressed in your first sample, but is either not expressed, or is expressed at a greatly reduced level in your second sample.
With respect to your conventional PCR:
So. Easiest fix is to simply order a different set of primers. I usually order two or three different primer pairs for each gene of interest, because primers are cheap and sometimes a given pair...simply doesn't work as well as it should.
Alternatively, you could play with your cycling conditions to try and enhance the specificity (higher annealing temperature, shorter extension time, etc). Primer dimers can often be avoided by a simple 1 degree increase in annealing temperature.
@John Hildyard, Many many thanks!!!!!!. Your suggestions helped me to have a new look on the problems I had stated.
It should be interesting to know the kind of mutation is present in the mutant. It is a single mutation (change of a codon), an insertion of a gene of resistance to an antibiotic, or a spontaneous mutation.. In anyway a RT-PCR should be stopped at 20 cycles and it is better to try less than 20 cycles. Did you sequence the parasite "c-DNA"?
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