I plan on using the Pfaffl relative quantification method to check for changes in gene expression and determined primer efficiencies (using a standard curve of cDNA dilutions) with 500 nM primer concentration. Do I need to use the same primer concentration in my experiment comparing different treatment samples?
As a rule, when you do experiment on set of samples you should keep the conditions as similar as possible. Otherwise there is a possibility that your results will be affected by unaccounted factors of condition variation.
As long as you determined optimal primer concentration by test runs you should follow it throughout your whole experiment.
As a rule, when you do experiment on set of samples you should keep the conditions as similar as possible. Otherwise there is a possibility that your results will be affected by unaccounted factors of condition variation.
As long as you determined optimal primer concentration by test runs you should follow it throughout your whole experiment.
I agree to Alexandrs` comment. Moreover, yes the primer expression may affect the reaction (higher conc. may lead to inhibitory/efficacy reducing effects in this context). An optimal protocol should be generated following standard protocolls (see relevant literature).
Yes, You should always use the same concentration of primers for all your samples/genes in one experiment. Otherwise, you are adding more factors of possible variations. I would recommend that when you are performing optimization of the entire qrt-PCR for a new set of samples/new target genes (temperatures, the concentration of primers, polymerase, concentration of cDNA, magnesium etc.) - always optimize on your samples or genes that cause you the most trouble (for example the worst conditions of an expousure experiment, the gene which is the least expressed etc), instead of optimizing on only control samples and stable house-keeping genes. It will save you a lot of time.
Yes, primer concentration does influence qPCR efficiency. In fact, adjusting the primer concentration is one trouble-shooting step to optimize efficiency.
Hi Gillen, I am just wondering how did you judge primer efficiency for qPCR? Did the melt-curve gave single peak or multiple peaks? Also did you check the amplicon size thru agarose gel electrophoresis? Getting single band in the gel will give you single peak in the melt curve.
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