I`m wondering about cDNA dilution and primers concentration of target and housekeeping genes in qPCR (using Sybr green) assay. Let`s suppose the following: by testing GOI primer efficiency (in a standard curve experiment) I find that it has an efficiency between 90-110%, using a 1:2 dilution of cDNA and 600nM of each primer. Should the endogenous gene perform in the same conditions? Or can I use differente dilution factors for cDNA to quantify target and housekeeping genes in the same plate?
Anytime you want to analyze the expression of your GOI to a housekeeping gene, you have to have comparable efficiency to accurately normalize. I usually work with PCR efficiency that differ no more than 2% (e.g. my GOI is 99%, my housekeeping gene 97%). Depending on what software you use, the PCR efficiency may be displayed already, if not, look at your standard curve and do some efficiency correction by adjusting the slope so that slope of GOI and housekeeping gene are within 2%. There are limitations of course to adjust the slope, so optimizing the qPCR reaction itself is key.
What do you mean by 1:2 cDNA dilution? I typically reverse transcribe 1000ng RNA in a reaction volume of 20ul and use for qPCR 2ul (theoretically corresponding to 100ng).
It is always better to keep the dilution of your cDNA 1:10 ratio. Hope so your CT value doesn't show much variation between your gene of interest and housekeeping gene. You should use the same primer conc. for GOI and housekeeping gene also. You are using same cDNA for GOI and housekeeping gene. You can't use differente dilution factors for cDNA to quantify target and housekeeping genes in the same plate. You should use same dilution factor for both genes.
Hope it helps you.
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