I am getting some extra bumps in my qrtpcr for actin. I see it in the nyc and in the x10*5 standard. So I think is primer dimer forming when there is not enough template. I tried adding more cDNA but that didnt help much. Now I made more cDNA with more RNA but some of the samples are giving weird amplification curves now and other samples still give bumps.
Dear Adela Oliva Chavez
You need to share the qRT-PCR and melt cure graph and also the brief protocol you followed then only possible to help you
Best
Amit
https://www.slideshare.net/idtdna/troubleshooting-qpcr-what-are-my-amplification-curves-telling-me-33646672
Hi Adela
Like Amit said, it would be nice to see your melting curves. That way we can help you better!
Overall, a primer dimer effect occurs before your melting curve peak, and you will see a bump before the main peak. This can happen in your NTC but also in your samples, so the fact you have a bump in your ntc not necessarily means you have a primer dimer. When primer dimer occurs and you see a bump before your main peak, this means before your DNA is melted and amplified, your primers are randomly binding to that material, hence your primers will not be really suitable. On the opposite, if your "bumps" are after your melting curve, this means you possibly have a contamination. This means after your DNA is melted and amplified, you still have some stuff that are being bind to, possibly random material.
The fact that you see amplification in your NTC makes me thing you definitely have a contamination. Possibly from the water you are using, since I assume you used water for hydrolysing your primers. Your SYBR green can be contaminated to, you may want to think to make small alliquots and thaw only one per experiment.
Also, a few things to consider - have you performed a primer blast analysis of your primers? if you have multiple predictions, then you increase the risk of amplifying random sequences. Are you using your primers in the correct concentration? I use 10uM in the reaction, diluting from my 100 uM stocks. If you add more RNA or DNA, it does not matter for avoid the primer dimer. If you add more genetic material, then your primers will have more chances to bind to that unspecific material or to your contamination. It will be also nice to see if you have a good purity of rna by nanodrop. In addition, if you add more cDNA to your reaction, you will get the opposite of what you want. You will increase the amount of genetic material your SYBR green needs to amplify, and you will get artefact data, as you will be "pushing" your amplification above the efficiency of your SYBR.
Hope this makes sense, keep us updated and then we can give you further tips!
Noe
Thank for your answers. This is a picture from the melting curves of the standards and the NTC. As the standard is reduce that second peak appears and it is the same as the NTC.
These are the melting curve of the same samples with a different set of primers.
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