Primer Efficiency (how to change the slope) - (Mar/07/2007 )
Hi guys !
I'm new to qRT-PCR (first week...) and I have a question (i.e. problem...).
I did a standard curve with 15,10,5,0 ng of cDNA with different sets of primers.
The slopes were different between the genes analyzed. Some slopes were -3.0 to -3.6, but other gave a slope of -2.8 or -5.4.
What can I do in order to calibrate the slopes ? Should I change primer concentration? cDNA amount ? Or must I design other primers and hope for the best ?
How can I determine the optimum cDNA amount ?
I'm using the ABI 7700 and ABgene syber kit
Let me see if i get it: so uoy tried different cDNA quantities? Does your syber kit mentioned the right amount of cDNA to use? For the slope you should use diluitions series of cDNA... to check what dilution fits well for each set of primers and to check the primers efficiency.
I hope these informations are helpfull for you!!
well, first of all, how are your dissociation curves? if you're getting a single product, don't toss out your primers just yet efficiency can be fixed
I would set up a titration grid, changing concentration of both template and primers. you will find a range of cDNA concentration at a given primer concentration will give you good efficiency, which will give reliable results
THNAKS aimikins !
The dissociation curves are great (single pick, Tm as expected). I'm trying to decide on cDNA/primers concentration - and that's a mess. To make matters even worse I started testing two kits of SYBR (ABgene and Finnzymes’ novel DyNAmo™ Flash SYBR) - both give me the same Ct, but the recommended primer concentration for each are very different (50-300nM and 300-1000nM, respectively), I'm currently working with 10ng of cDNA (ct for GAPDH is at about 16, and for my other genes at 19-24 cycles).
so in short my problems are -
1. I'm not sure how to choose a kit to work with
2. how to determine my cDNA concetration ? (can it differ between the analyzed genes and HK gene) ?
3. are there any guidelines to follow (i.e. "to lower the slope use higher primer concetration")
4. when do u decide that your primers are not good enough and you need to use a different set ?
Can you recommend such a grid to work with ? (if understand it correctly it may be over 50 reaction only for calibration)
well, yes, initially it will take many reactions to calibrate. unfortunately, if you've tried the 'stab-in-the-dark' method and not found your optimal concentration, it is necessary to fine-tune. I'm sorry!
as far as which kit, it's really up to you. I haven't used either myself; perhaps someone else here can give a comment? which one is easier? which one gives cleaner, more reproducible results? I don't know that it matters, unless there's a flaw in one kit or people find that it gives flaky results over time. Perhaps see if they're both published with equal frequency, and if they are go for the cheaper one I wouldn't spend too much time mucking about with several kits, but it's up to you.
I have generally found that less primer is better, but it's different for every gene and you'll just have to find it empirically.
I think, if at all possible, that it's wise to use the same cDNA concentration for your sample and control standards. likewise, whichever kit you use, try to use the same kit for all the experiments you will do. there are so many picky little parameters that can affect the outcome of these reactions, the more things you mess with the higher the potential for error later.
we don't care here if the cDNA is at 5 or 50 ng, it's the efficiency that's key...likewise with the difference in Ct between controls and samples. as long as it's reproducible and the efficiency is good, anything in the 12-30 range is OK...there are differing opinions here; you may wish to read over old threads. what I mean is, in my opinion, if your control-GAPDH Ct is usually ~25 and your sample Ct is usually ~18 if you run a given amount of cDNA, I don't think that is a difference you need to worry over, as long as both amplification reactions have efficiencies close to 100 and close to each other, do you see?
as far as throwing away a primer set? well, if I find that a given concentration range for cDNA gives good amplification efficiency and clean curves with 4 out of 5 primer sets, I'll chuck the 5th and try for a re-design after thorough testing of variable primer concentrations. sometimes you just have to do the best you can, but suboptimal primers will not give you good results from one experiment to the next, and no amount of wishing will change it. I really hope you have an easy time finding your optimal concentrations, but it is what it is...and as soon as you optimize, the reactions will run like clockwork afterwards, however much of a pain they are in initial setup
Hi all and especially aimikins (a.k.a. lifesaver)
Thank you SO much for your adivse. a little update -i have chosen ABgene's Kit (though both of them were very good,AB gave lower stdver).
a little primer calibration and my slopes are -3.48 (GAPDH) and -3.27 (target gene) - so I'm quite happy !!
I'm still not sure how much cDNA template to use, working with 2-5ng sounds a bit ridiculous (when working with RT-PCR i usually used 25ng/reaction) i think i should stick with my 10ng... but i really don't have a good reason for that... but if I understand aimikins correctly as long as my Ct, are stable at 12-30 cycles I'm good.