# HELP! confused with validation of efficiencies - (Mar/15/2006 )

Hello,

I'm trying to figure out the efficiencies for my target gene and housekeeping so that I can compare using the ddCt method. I'm using a standard curve of 2x dilutions from 50ng to .78ng for target and housekeeping. I'm using the Roche LightCycler 2.0

I'm very confused about which graph/slope I should use to figure this out. Here's what I've calculated:

using the Cycle number vs. log of initial concentration; Efficiency=10^(-1/slope)
slope for target= -3.568; E= 1.9066
slope for hsk= -3.333; E= 1.9954
-- if 100% efficiency is 2, then target efficiency is 95.33% and hsk efficincy is 99.77%
since it's within the 5% range, this would mean that I can use the ddCt method, correct?

so then I used dCt vs. log initial conc; absolute value of slope <0.1
I subtracted Ct(target)-Ct(hsk) to get the dCt
This graph is supposed to give me a horizontal line, but it didn't. the absoute slope is 0.23
this would indicate that the efficiencied aren't the same.

can anyone tell me which formula i should use to trust my data? the customer service of roche told me that I should follow the Cycle number vs log conc since it's provided with their software.

here's another observation: the slope for hsk gene using the Cycle# vs log graph is different from experiment to experiment. What does this mean? pipeting error? degradation? I have to validate 6 diff genes and the LightCycler only has 32 wells so I have to use 7 standards+blank in duplicates, so i have to run one target and hsk per run.

-el2173-

ok

go to ABI's website

do a search for "user bulletin #2" (I can't always get the link for that one to work, maybe because of the site's security? dunno)

anyhow, there is a chapter in there on validating ddCt. it will be like the bible for you and tell you how to set up your calculations

I hope this helps!

-aimikins-

QUOTE (aimikins @ Mar 15 2006, 05:45 PM)
ok

go to ABI's website

do a search for "user bulletin #2" (I can't always get the link for that one to work, maybe because of the site's security? dunno)

anyhow, there is a chapter in there on validating ddCt. it will be like the bible for you and tell you how to set up your calculations

I hope this helps!

thanks for your reply. i did look at that manual, which is why I'm confused right now... they prefer to graph dCt vs log and take the slope. Roche looks at the slope from Ct vs. log graph for each gene and compares them. I guess the difference between the methods is that one normalizes to hsk gene before figuring the efficiencies. I'm wondering which method is more precise.

-el2173-