Cells were transduced with a shRNA lenti particle to knockdown a particular gene. Instead of seeing a knockdown in gene expression I got a shift in my Tm from 81.83 (in both CTL untransduced cells and cells transduced with a control shRNA) to 85.43 (in cells transduced with knockdown vector) still with a single peak in each sample. Any idea what this means?
no changes in the CT values?
Have you look at the proteins to see if your shRNA treatment affected the protein levels of your target?
Have you tried different sets of primers for your qPCR?
No changes in CT, Finishing western and looking at different primers today. But I would like to know what can cause a shift likt that.
With the data at hand it's difficult to have a theory... A low affinity off target for your primers? but it would be surprising since you have the exact same Ct values... what are the average Ct value for your target? Are you using SYBRgreen or Taqman?
Have you tried running your qPCR products on a highly concentrated 1.2-1.5 agarose gel to see if there is a difference between your amplicons?
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