In the primer validation, with the serial dilution of the cDNA, I have a melt curve with a single peak. However, when I do the gene expression using control and treatment samples (I am evaluating the effect of environmental contaminants, so I have tissue samples that have not been exposed to the contaminant and exposed samples), the dissociation curve is as shown in the attached image. What does that mean?
best regards
Hi Samara, how are you doing today?
Well, multiple peaks in the melt curve could be several things. The first case is the co-amplification of genomic DNA and your cDNA sample. Your cDNA sample has genomic DNA mixed. Second could be primers dimers, your primer hybridizes with itself and generate a small amplicon. Third is off-targets amplicons, your primer binds a different region that was originally design, and the last could be a high guanine-cytosine content, which can cause a different peak in the melting curve.
However, analyzing the picture, the curves are too close to be a primer dimer or off target amplicon. Your problem could be just an DNA contaminant or high guanine-cytosine content in your target gene. To avoid genomic DNA amplification, you need to design primers in exons flanking an intron with at least 500bp. The polymerase of qRT-PCR mix has no efficiency to amplified this amplicon size. High guanine-cytosine content in your gene is not necessarily a problem, and depends of the sequence, length and heterozygosity of your gene or the kind of model you using. Well, my best regards and good luck with your research.
Hello Samara
it could be due to genetic polymorphism or alternative splicing or just a technical issue .
Hi,
I agree with the comments. I would suggest polymorphism in your target sequence or an evaporation of liquid in some wells leading to higher salt concentrations and shifted peaks. Have you repeated the analysis? Are the same samples showing the shift? Are the two samples with the most left-shifted peak form the same individual?
Good luck,
Norman
Hello Samara Silva de Souza ,
Heterozygous nature of the target gene might be affected for the different melting peak of your sample. so always better to avoid the polymorophism when designing the primers. Also, i would think some technical errors.
Have you tried running your samples out on an agarose gel? I'm assuming you'll see only 1 band, but it's good to check. As other said, try sequencing a few of the most extreme left & most extreme right. With such small products you can see the influence of one SNP or a tiny InDel.
Good luck!
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