Hi All
If you take a look at the image, the upper virus X assay (for Rotavirus) is giving off a lot of fluorescence at the end of the run (circled) which is clearly not real (we know these are Rota negative samples), this is affecting the baseline setting making the positive samples look like they are giving off poor signal.
The lower figure for the virus Y assay (for Norovirus) shows what the assay should look like but is just an example targeting a different virus
Is this primer/probe degradation and if so why don`t I ever see this is any of my other assays
Many Thanks
MArk
Hi Mark!
I would say it is not degradation, but probably primer dimers. Is it the first time you use this primer combination for qPCR? A way to test if it is really primer dimers is to make the melting curve... you know, different products will have different Tm. Another option, in case your qPCR machine does not allow to do that directly, is to take the qPCR product and run it on an agarose gel. You may see two bands for the positive samples (or not,maybe just the specific one)and one for the negative ones, different from the specific expected size. I guess you are using sybr green? If so, indeed primer degradation may cause primer dimers, in case you have not seen that in previous experiments with the same primers. If this is the case, just order new ones. In case it is the first qPCR you do with these primers, design new ones for rotavirus. Another option(a dirty one, but still, if the results are very urgent...) is to run less PCR cycles, so that this unspecific amplification does not occur.
Good luck!
Hi Victor
We are using TaqMan probes (FAM-BHQ). We have repeated the test with different samples and seem to get the same result.
I will run it on a gel and see what we get
Many Thanks
Mark
mmmm, if using taqman probes, the primer dimer formation is unlikely...well, it is unlikely to produce a signal, although they may decrease the efficiency of amplification. Is there any possibility that your samples have been cross-contaminated? meaning, that your positive samples have contaminated the negative ones? Are the rotavirus-negative samples positive for any other virus, so you can check if the rotavirus-positive samples show any signal for these other viruses?
This is not specific amplification. Primers look for bait - and just finally hit something. Where have you been??
I agree that it might be contamination. Since you've tested different samples, the contamination could be in your master mix. Once an under bachelor student from my lab accidentally used a dirty pipette tip and we had to open a new master mix.
Have you optimised the Rotavirus assay (i.e. run a primer concentration experiments, adjusted the probe concentration)? The positive plots look a bit linear for my liking. This may not get rid of your problem but, if it were me, I would be happier exploring the underlying issues if I knew this were the case
Dear Mark,
did you manage to solve your problem? if yes what was the problem? because i am experimenting the same issue. It is not a primer / master mix contamination. Yes reducing the number of cyce reduces the aspecific amplification but it is not optimal as we have a standard curve and the final dilutions won't appear if i reduce the number of cycles. How can it happen if it is primer dimer? why should it look we have an amplification?? thank you all for your help !!
Hi Mark,
Did you find the reason for the problem?
I experienced absolutely same problem. Even I come up to the moment to open new master mix, water, tips for pipettes and run PCR for only water without any positive samples on the plate. So, the unspecific fluorescence after 35 cycle appears again. Just like the first picture.
I try to establish assay for virus possessing high AT reach genome (I cannot mention the virus because of company politics). It is very hard to find enough long region for primers and probe selection/design.
One of the problem I realized is long primers because of high AT reach genome. I thinking for shorter primers with nucleotide substitution of C to pdC and T to pdU to increase Tm. But, the shorter primers will be reason for unspecific amplifications or not!!! The virus will be looked in samples with high number of species.
I will very appreciate any suggestions and comments about my ideas for solving the problem!
Kind regards!
Dimitar Yanev
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