Optimizing primers for SYBR green qPCR? - Optimizing primers for SYBR green qPCR? (Oct/26/2005 )
I am new to qPCR and have two questions. I have been reading a few protocols that are posted online in addition to the protocols from Perkin-Elmer. PE suggests that every set of primers must be optimized before use. However, I have read a couple of other protocols that state to use primers at 0.2 micromolar and optimize only if there are problems. It seems that the PE protocol uses a lot of reagents (good for them $$) while the other protocols may minimize the expense. Since we are a poor lab we would like to minimize our cost.
Anyone have any suggestions regarding this? Do I need to do all the optimization controls as suggested by PE?
I have a second question, we are using a PE 7700 machine. Does it matter whether I use 25 vs. 50 microlitre reactions?
can't answer the 25vs50...I use the ABI 7700 and I find that it doesn't matter
about your primer optimization. to start, I don't think you have to do 6 plates of optimization for each primer. after a lot of trial and error (and too much $$ to ABI ) when I get in a new set of primers, this is what I do:
set up some dilutions around the amount of template you will normally use (this differs for everyone unfortunately and you do have to figure it out the hard way based on cell type, RNA yield, spec characteristics in particular).
I do it this way, assuming 1X template is about how much I will usually add for all my genes and 1X primer starting at 2 uM, each one in triplicate:
4X template / 1.5X primer
2X template / 1.5X primer
1X template / 1.5X primer
.5X template / 1.5X primer
.25X template / 1.5X primer
and so on, repeated with primers at 1X and 0.5X (this uses less than 1/2 a plate, with NTC wells and appropriate controls) usually I get good reaction efficiency at the 1X primer with template across the range, that tells me I can good results if my expression changes because of my experiments.
If I don't get good efficiency, I can fine-tune from there. For example, if the 0.5X primer has awful results, but the 1X and 1.5X are not too far off (but still not ideal), I can add some finer dilutions between the two to narrow it down. I have NEVER had to add different amounts of either primer, but you are supposed to set up a plate like that if you do everything the manufacturer's say you need to. what a waste! I will tell you that I use 7 primer sets (7 different genes) and have only found 2 that require the use of different amount of primer than 2 uM (and both needed 2.5 uM; a small difference but the change in efficiency was key in order to validate the ddCt method)
by the way, save TONS of money and time...put a little extra effort in now and set up for the ddCt method. You won't waste a bunch of every plate making standard curves, if you are doing a whole bunch of screening for a handful of genes (I am assuming this is your plan since you use SYBR green and you want to keep it on the cheap)
I think for optimization, you can probably get away with doing things in duplicate. IN addition, most people are using 25ul reactions and most of the newer machine are now able to do 10ul reactions. Definitely a good way to save money. In general, with SYBR green, I find it does pay to do the optimization due to the sensitivity of primer dimers in the reaction. You will definitely find differences when you run the melting curve or run the products out on a gel.
We routinely run 15ul rxns on the 7700. We've run 1,000s of rxns...cuts the cost significantly.
Specific primer design is again a mandatory requirement for lowering the total assay cost.
My experience is that optimizing primers is usually not cost-effective. After optimizing lots of sets, my strategy nowadays is: order primers, put some time in designing them. Try them on a dilution series of the sample I want to analyze using a standard protocol (minor modifications allowed, i.e. annealing temperature or slightly different extension time depending on lenghth of the amplicon), always using the same primer concentration (0.5uM). Usually, these works with the big majority of primers, if it's almost there but not quite, might play a bit with the annealing temperature. If first time looks very wrong, then I order new primers: much cheaper than wasting reagents trying to optimize...
So basically, I think what the companies advise about optimization is just rubbish!
Amen to Erica. Just redesign the primer if you have trouble.
a second amen to Erica.
we had a company tell us to optimise the individual primer separately (!!), and yeah, money grabbing rubbish.
design a good set, test them out on a plain RT PCR (and run on a gradient PCR if things don't look exactly the way i want, saves time).
i actually use a 12.5 ul reaction (in triplicate) for actual real time.