Real time PCR - Standard curve construction (Apr/10/2010 )
I am a beginner to Real time PCR and i have to perform both absolute and relative quantification methods. How is the standard curve generated? should i use the serial dilutions of genomic DNA, or cloned plasmid or the cDNA?. is there any protocol for this in the web?
and i have to perform an expt to determine the copy number of a transcript. i would like to know if there is any protocol for this.
if the template is cDNA then i would use in vitro transcribed and then reverse transcribed RNA as absolute standard. but if you are a novice to real time PCR i recommend you to have a look at the literature thread (in the sticky section of this subforum).
When performing real time PCR, one important rule of thumb is to prepare the template you will use for your standard curve in the same way you prepared the template of your unknown samples. So if your samples are tissues that you used to extract RNA from and then make cDNA from, then your standard curve samples should have been prepared in the same manner.
A standard curve is nothing more than a serial dilution of a control sample of known concentration. For example let's say you made cDNA from 1 ug of RNA. You would then go ahead and dilute that cDNA 1:10, and then dilute the 1:10 dilution 1:10 again. Continue doing that until you have the following dilutions: 1. undiluted cDNA, 2. 1:10 dilution of original cDNA, 3. 1:10 dilution of 1:10 dilution (aka 1:100 dilution), 4. 1:10 dilution of 1:100 dilution (aka 1:1000 dilution), and 5. 1:10 dilution of 1:1000 dilution (aka 1:10000 dilution). Run real time PCR experiments (using your assay of interest) using each one of these templates (at least in duplicate in each case) and then perform a linear regression of these data points (most real time PCR software packages do this automatically). Done, you have a standard curve.