I am a new to q-PCR. I used sybr to detect some gene expression. The result is not good, and I found my melt curve is like this. I read some troubleshooting, but I cannot find the answer. Can you help me?
It seems your primers amplified at least two separate products. If you still have the plate from your qPCR you can run the PCR products on an agarose gel and verify that there are two bands.
There are several reasons this could occur. It could be that your gene of interest has different splice variants and you have amplified both of them. It could be that your primers are not specific enough for your gene of interest and you have amplified another gene as well. It could be that you have both genomic DNA and cDNA in your sample and you've amplified different products from the different templates. (Primers for qPCR are supposed to be designed across the exon junction to decrease the possibility of this happening even when the sample includes some genomic DNA contamination).
You may want to take another bioinformatic look at your primers and see if you can determine whether they may be producing off-target products.
Hey,
For me it looks like you have 2 different PCR products.
Check again your primer pairs (maybe they also bind somewhere else).
if it is cDNA make sure that it is not because of splicing variants (if your primers are over more then 2 exons.) Or also that you detect non spliced and spliced products.
Best situation would be if you use 2 exons and one primer binds half in the one exon and the other half in the other exon. By doing this you prevent getting and analyzing non spliced RNA.
you should also run (when you redesign your primer) a temperature gradient.
Did the primer ever worked before or is it a new one?
all the best,
Sebastian
It seems your primers amplified at least two separate products. If you still have the plate from your qPCR you can run the PCR products on an agarose gel and verify that there are two bands.
There are several reasons this could occur. It could be that your gene of interest has different splice variants and you have amplified both of them. It could be that your primers are not specific enough for your gene of interest and you have amplified another gene as well. It could be that you have both genomic DNA and cDNA in your sample and you've amplified different products from the different templates. (Primers for qPCR are supposed to be designed across the exon junction to decrease the possibility of this happening even when the sample includes some genomic DNA contamination).
You may want to take another bioinformatic look at your primers and see if you can determine whether they may be producing off-target products.
How do you run your qPCR? One step or two step?
Have you run these samples on an agarose gel?
This looks pretty much like you have more than one product, which could mean unspecific amplification, sequence variants (different allels) or splice variants.
Dear Xie: First which curve you are talking about.. generally it looks like multiple products and also what is your tm??? Because it looks like if you increase the Tm the curve may look better, but anyways you can try with other set of primers.. and also check the cDNA quality.
I agree with previous answers. This looks like of two different PCR products from the same set of primers. Check this PCR in an agarose gel, but I think you will end designing new and more specific primers.
If you have two sets of primers in your plate, then those are two melting curves corresponding to two different amplicons. If you have only one set of primers, then the situation is as follows:
-Left curve (Tm=72°C ca.): you have amplification of a dominant product (presumably your cDNA) and, in addition, of a less represented product (presumably genomic DNA);
-Right curve (Tm=80°C ca.): genomic DNA amplification occurs.
Another possibility, although the peak is really high, is that the left peak corresponds to primer dimers while the right peak to the correct amplicon. But again, this seems quite difficult to imagine.
- Left curve: shorter splice variant; Right curve: longer splice variant.
How was your RNA? Clean? Was genomic DNA present in your samples? Did you amplify a housekeeping gene and, if so, is the amount of this gene lower in the samples which give you the peak at 80°C only?
I am totally agree with previous answers. This looks like of two different PCR products from the same set of primers. I think you will end designing new and more specific primers. You can design your primer with this option "Primer pair must be separated by at least one intron on the corresponding genomic DNA" to be sure that you don't amplified genomic DNA.
If you can re-design the primers with higher Tm, in order to avoid the undesirable products? Did you run the PCR products on agarose gel? It will gives you a quick answer, about the primers and the products.
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