I performed a qRT-PCR reaction for the CDC20 gene on drug-treated cells and non-drug treated cells and obtained these melting plots and I am just wondering how to explain the shoulders on the end. Would these be indicative of non-specific primer binding? And if so, could that have to do with the volume of primer added (a pipetting issue)? Not everyone in this laboratory course had the same issue, and I am trying to explain my results
Difficult to say. It could be a smear of unspecific products or the result of a temperature-dependent quench effect (fluorescence decreases with increasing temperature [that's a physical effect, independent of melting]).
Dear Anouk Schrama , if you want to estimate drug effect first of all you have to consider Ct in treated and control samples. Regarding melting peaks shifting, it is ok. It seems non homogeneous but even 2 degrees it is not a big trouble. In the case of HRM - yes you can concider it like for example, SNP, but with SYBR Green for me it is not significant. At least one degree.
If you want to be sure in right product - Sanger sequencing can help you.
Moreover you can check the results in agarose gel.
For such short products a usually amplified in qPCR (80-150 bp, more or less), a 6% PAGE is much better than an agarose gel. Capillary electrophoressis (e.g. Bioanalyzer) has a high resolution, too, but not everyone has access to such instruments.
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