Delta-delta Ct method and PCR efficiencies-Real-Time PCR

Hi all,

I have a dilemma - I've run a dilution series on my housekeeping gene and gene of interest to test for equal primer efficiencies, graphing Delta-Ct vs. log concentration, and my primers pass the test, with a slope much less than.1. However, when I graph the standard curves for each primer pair, the slopes are not within the -3.2 to -3.7 range to indicate good efficiency (they are -2.88 for gene of interest and -3.13 for housekeeping). How 'bad' are these efficiencies and does it matter as long as they pass the equal-efficiency test? (I've gone through all the tweaking of the baseline, threashold, and casting out outliers and these are the best slopes I get!).

By the way, what does an efficiency value over 100% mean??

I was told by a veteran...who was my own jedi-master when I was a young qPCR-er...that if your efficiency is greater than 85%, you may use the standard curve method and expect reproducibility...if your efficiency is between 90-105% you may use the delta-delta Ct method, as long as your GOI's are within 5% of each other..and that any efficiency over 110% is an imaginary number and your conditions need to be changed (this is not more efficient, it is poor efficiency)

thank you, aimikins. Looks like more troubleshooting then for me.

May the force be with me

have you been following that other qPCR thread today? I've been having some similar discussions with someone else.

anyways, it may behoove you to try a different template range, and what about adjusting primer concentration? you have probably already tried this

You could still use ddCt method with unequal efficiencies, you just have to take it into account. For example use REST qPCR program from Pfaffl.

Well, not sure which thread you mean, but I did read (I think it may have been your post) yesterday about narrowing the range of template concentrations for the standard curve. I'm curious, though, how this can be extrapolated to the actual experiments. If one can't get good efficiencies with a broad-range standard curve, will it accurately measure expression in tissues with low expression? This is a key point for me because I expect my GOI to be very tissue specific.

I did alread optimize for primer concentration.

Another wrench in the works is that it appears that our system may need re-calibrating; I found some background in some wells, so will probably re-run the entire test experiment to see if I can't get better efficiencies.

Thanks for the help!

QUOTE(aimikins @ Dec 7 2005, 06:35 PM) [snapback]33831[/snapback]

Excentrifuge (or anyone?),

If you want to use the ddCt method with unequal efficiencies, does that mean you must run an efficiency curve with every experiment? Or, do you do several experiments to determine efficiencies in your system and then just assume they do not change from then on?

My problem is that I don't have one set system. I will be doing RT PCR on freshly isolated tissue from different human donors each time. ABI told me that efficiencies vary from tissue to tissue, so I can't assume they are always the same. Does anyone have an opinion on that?

Thanks!

we use only primary cells from one tissue type; our GOI's expression very rarely changes more than a log. I cannot answer that question, although I suspect if you are expecting a very large range in expression that you may have to use the standard curve method if you want good accuracy. We only change the way the cells are treated prior to harvest for RNA, and we tested a couple lots with our controls to make sure the expression changes were fairly constant with our treatments...and we run the same controls every time we grow up a new lot of cells. But, if you were looking at large changes in several tissues, I would imagine that accuracy and reproducibility are even harder to achieve.

Actually, your response is very encouraging to me! I'll also be using primary cells from the same tissue type. But, as you know, my samples come from humans. I suspect that samples that come from the same strain of rats or mice might be more similar than from various human donors...is that what you use? But, now I know that it is worth investigating. Thanks.

we use HNEK cells...human neonatal epidermal keratinocytes. each lot was pooled from three separate neonatal donors. they usually try to get at least two ethnic groups involved, so the samples are really pretty varied...but not so much as they would be from adults. We look at factors involved in innate immunity and pathogenesis of infection. I would think adult samples would typically show more variation, but I'm not really sure. we use the cells within 4 or 5 passages out of the cryotube, then get another lot.

I really suspect it depends on your GOI's. If I had seen larger variations and was unable to get good efficiency, I would have had to try a different approach.