I have been trying to validate a qPCR reaction, where the expected amplicon is 98 bp long. Initially, I made standard dilutions from qualitative PCR product. However, when I ran it the first few times, I got a higher Tm peak in the melt curve analysis (around 84-85 degrees) than the actual samples. I ran the qPCR products on agarose gel, and found that there were 2 bands, one much larger than the target amplicon. I then cut out the target band from the gel, and prepared dilutions from that to exclude the higher amplicon. However, I am again getting a melting peak at much higher temperature (again around 84-85 degrees) than the samples, which have consistently been showing a peak around 75-78 degrees, but in some of them, a very small peak is present around 84-85 degrees as well. This makes me think whether I am actually looking at the right melting peak (75-78 versus 84-85?) or is it something else? I'd really appreciate if I could access some source that predicts reliably the melting temperature for the amplicon. I tried uMelt, and it also shows Tm of 84-85, so I am very confused now about how to proceed, whether improving the standard dilutions somehow or re-analysing the target amplicon?
Hi,
as you did the gel what is the size of the two products in relation to the predicted one? In addition is it a mRNA specific primer pair? If so could it be amplification of gDNA giving the 2nd band? Further how strongly do you dilute your PCR fragment to be used? Sometimes if you have very early Cqs and your primers are not perfect they might lead to dimerization and unspecific amplicons. You could therefore check with a gradient if this is true here
Sven
Hi! I think you can try to first adjust the PCR conditions to avoid the amplification of unspecific amplicons. Sometimes when you have many template it is more likely to amplify unspecific products, so you can try first to use less amount of cDNA and check if you have a single peak in your melting curve and you can see in the gel if it is the size you expect. Also, can happen that the primers are not specific enough, so you can try to make a better design. You can make a multiple alignment with similar sequences and identify primers with greater specificity. Dont bother too much about this melting temperature prediction you mention... To make a correct estimation of gene expression you should have a single peak/amplicon and you can confirm the size in the gel, when you still have two amplicons then you have to find a way to solve it. Hope this helps.
75-78 degree is typical melting temperature of primer dimers. Whereas 84-85 would be probable specific Tm of your desired amplicons.
Hi Megha,
You can use https://www.thermofisher.com/jp/ja/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html
or alternatively I would suggest running a gradient PCR to identify the best Tm for your primers and make sure your primers are just amplifying only your gene of interest.
Best!
Sven Reister one of the bands in the gel is the predicted size, i.e., 98 bp and shows up around the 100 bp mark. The other was around 430-450 bp or so...which seems to be genomic DNA contamination, as the primers are designed about that space apart between two exons. So yes, it seems gDNA there.
As for dilutions, I make them like 10-2, -3, -4, and -5.
Thank you very much for your reply! :)
Diana Duarte-Delgado Thanks for your help. I will try using less cDNA. I even doubled the time for DNAse digestion while extracting total RNA, but again got same results.
Yashwant Chavan that is exactly my doubt too. Thanks for your reply!
Pushkal Sinduvadi Ramesh Thank you! It seems that gradient PCR is the quite common suggestion here. I will definitely try doing that and hopefully my primers work.
You probably have your melt temperature figured out by now, but I also thought I would add a useful resource for anyone that has this problem in the future. I find this melt curve predictor to be really helpful because it helps idenity biphasic melt curves: https://www.dna.utah.edu/umelt/umelt.html
Amy Replogle Yes, I think I have figured it out. However, I have not still been able to validate the standard dilutions, :(. Thanks a lot for your reply, I indeed used uMelt.
- Badges
- Science method