different Ct values for RNaseP gene in human genomic DNA - (Jul/28/2021 )

Hi all,

I input 50ng/well of human genomic DNA to amply RNaseP gene.  I use primer-probe sets  which are commercially available.

To my surprise, I get  2-5 Ct differences between samples.

Any comments?

Thank you very much in advance for the discussion.

Alex

How did you quantify the DNA? if by spectroscopy (e.g. Nanodrop), the natural variation seen in the ratiometric values used for nucleic acid concentration are what might account for your variation. Otherwise it could be variation in your sample preparation, pipetting inaccuracies, reaction efficiency etc. There's also the possibility of natural duplication in genes/DNA content that happens in some cancer-derived cell lines.