Hello friends
I am trying to estimate gene copy index by using B-actin as a house keeping gene. I am getting 100% efficiency for my desired gene but failing to get for B-actin. Can any one give me idea how we can troubleshoot it.
Thank you
did you considder changing your primer annealing temperature?
if it's too high you'l lose efficiency, so I'd try lowering it (unless you have primer dimer problems?)
if you don't get your effiency up, you can always determine the PCR efficiency and incorporate it into your gene expression calculation.
The calculation normally includes a 2^n step where n is the Cq value. 2 is the value for 100% PCR effiency, but you can substitute this for a lower value in the calculation of the housekeeping gene.
Try diluting your cDNA template or redesigning your primers. The Pfaffl equation adjusts for less than 100% efficiency. http://relative.gene-quantification.info/
Actually I am using genomic DNA isolated from viral tranfected CHO cell lines. I did at lower temperature also at both 58 and 56 as suggested by Arnoud Boot. Still efficiency is very less.
it's no problem, just use the pfaffl method, and change the 2 to whatever your PCR efficiency is.
To determine PCR efficiency, just make a dilution series, and calculate from there using the Ct values.
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