Can I use real time PCR primers for semi-quantitative PCR to study specific gene expression? Will it give the expression pattern more or less similar to the real time PCR results? Please clarify my doubt. Thanks in advance.
If your primers are nascent and not labelled; in other words your qPCR was performed with sybr green then in principle yes you can. However there are one or two cautionary caveats to keep in mind
Im guessing your primer concentrations for Sybr green real time qPCR approximated to between 1 and 5uM @1ul per reaction. For semi quantitative PCR you should aim to use approx 10uM primers (1-2ul per reaction) on account of the lower sensitivity of gel based RT PCR
Also to discern accurate expression differences between genes you need to be sampling your products in with correct dynamic range. In other words where PCR is in the exponential phase of Amplification and not during stationary phase ( where signal is saturated or reaction rate limited and differences might not be distinguishable). This could involve setting up 3 sets of PCRs with a 5 cycle difference; say at 20; 25 and 30 cycles. It is likely one cycle number will be more optimal for distinguishing expression differences. In the case of real time qPCR the technology monitors amplification in real time (rather than end point sampling) which is why the Ct can be selected to be optimal
Apart from working blind in terms of end point conventional PCR also keep in mind that qPCR by virtue of detection method is about 10 x more sensitive than gel based PCR using Sybr dyes or ethidium bromide. Thus below 20ng it is difficult to accurately quantitate a band on a gel. This has implications for low copy number genes where Ct is > 30 cycles and might not be detectable by gel based PCR. I have experienced that situation
Furthermore if expression differences are > 10 cycles by real time PCR that might be difficult to replicate by gel assay
Nevertheless I have successfully mirrored qPCR results in a gel as described so it is worth attempting unless your GOI Ct is > 30 cycles
Finally for sharp bands @ sizes normally used in qPCR - that is 100-200bo - for sharp bands faciliting better quantitation I would and routinely do use 2-3% agarose gels
Why not, I am sure it will be amplifying in semi quantitative PCR, if your product size will be coming below 200 bp and above 100 bp you can use 1.5% agarose gel. if your amplification product below 100bp you can use 2 % agarose gel,to keep maintain amplification for 40 cycles..
yes you can use. u can also find perfect bp of your designed primers. before real time pcr u must go for semi quantitative pcr to verify your product size as primary screening.