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problem: low efficiency - (Aug/24/2007 )

I'm doing a relative quantification on several cell lines. I managed to do very good standard curves in both house keeping gene and gene of interest and when I started analyizing the rest of my samples, the slope of my gene of interest is bad while the slope of the housekeeping gene is mostly OK. Also the efficiency is very low, around 30,40%.
Does aybody have an idea why and what can influence the efficiency?

btw, I'm using sybr green and melting curves look OK


what about your CTs? if they are higher than 30 cycles maybe that's the problem and the slope of your dilutions won't work. Maybe the problem is that your gene is simply not being expressed yet huh.gif. On those cases I've used the deltaCT formula to compare gene expression.

Good luck smile.gif


My Cts are between 18 and 25 so this should not be the problem, there must be something else