I might be wrong but it smells like off target or any other unwanted amplicon. Have you calculated amplification efficiency for these primers? Values other than 90-100% might suggest that there's something wrong going on. I also suggest running the reaction on agarose gel suitable for your product length. If you use a dye that migrates during electrophoresis (like ethidium bromide) consider staining the gel after the electrophoresis - it's all to see possible other bands that might be a product of your qPCR. These are my standard tests for new primers sets for any qPCR, before starting measurements of gene expression. Unfortunately I don't know anything about CD34 expression to help you out, but hope you will find a solution to your problem. Best.

Hi,

The reaction needs little bit standardization in my view. As there are less CD34 templates the unused primers tend to show these extra peak. You may try concentrating your sample or add little bit more template. Your present situation is like looking into the graph with a magnifying glass as the scale is really low.Once you get a good peak for CD34 the smaller once will disappear. 

Hi,

Try to optimize your primers condition or as Iftikar said it may be because of low templates concentration and add more templates. Best.

Iftikhar and Sima, thank you both , but actually I am running 6 genes in parallel in addition to the house keeping gene primers, all show no such abnormality. In addition this phenomenon seen even in high concentration of templates consistently in 3 cell lines.

Aditi, Thank you, I'll try your suggestion, however the question why this happened with CD34 primer only in comparison to the other. Do you think there any specificity for this primer in AML cell line? Thank you again

it seems  like u have multiple amplicons with these set of primers. however since the amplitude of the peak in melt curve corresponding to non specific amplicon is less u need to optimize template concentration along with annealing temperature. 

Have the primer sequences been blasted to ensure amplification of specific product? It obviously looks like off target amplification. If the sequences are binding to non-specific regions even optimising the conditions may not help too much and you may need to redesign new primers. 

I also think there are 3 thing to do here, hopefully 2;)

- increase the annealing temperature

- look for the right concentration (look into efficiency, like Paweł wrote)

- get new primers:)

I suggest qPCR primer banks.

And, obvious stuff but those get lost the easiest;) - what about other  primers (for nonhousekeeping genes), do they run right with these AML templates?

Yes Jakub, all other 5 non houskeeping primers and the housekeeping one worked pretty fine with the same template.

 Hi, I'm so sorry, I have tried three times but I am failed to download your document because of the internet problem, but from the discussion above, I want to tell my experience. As Pawel Olszewski mentioned, you'd better run a agarose gel, then you can clearly see whether the product is CD34. I don't know much about CD34 expression, but recently I detected the expression of another gene which is low expressed and the CT value is more than 35, the melt curve was also abnormal too. The results also had great differences between double-wells due to the high sensitivity of QPCR. I repeated several times, sometimes the melt curve seemed good, especially in high expressed tissues and samples. The results of housekeeping genes were pretty good as you said. The primers I used is referred from the published paper. I think there are several factors might influence the results of QPCR should be considered: concentration of template, the expression level of the gene (you'd better find a positive control), the reaction condition and the specificity of primers. I'm trying to adjust my test conditions and improve the quality of cDNA. GOOD LUCK.

Hi,

from the melting curve plot it seems that your primers are not so specific for your gene of interest. However the heights of your main peaks are not so high and this is probably due to the low expression of CD34. Maybe if you re-run the PCR with higher amount of DNA you could get a greater difference between the ''aspecific'' peak and the main peak. I'm suggesting this because maybe, with this pair of primers and with this amount of DNA, the efficiency of the reaction is not optimal. For this reason I also would suggest you to calibrate CD34 primers. As the other researchers said, run your PCR products on agarose gel; if your primers are specific you should see only one band on the gel. You can also check the specificity of your primers using primer-BLAST, but the agarose gel could be the best solution. In anycase, if you change primers, you should always calibrate your primers and check their specificity on agarose gel, at least one time, in order to show that your primers are good for the system that you are using in your  experiment. Sometimes some primers work perfectly with one cell line but they don't with another cell line. Best