what is the prerequisite before you conduct relative quantative real-time PCR?
In my opinion, it is always better to prepare a standard curve for every new set of primers that you use for real-time PCR. I think this holds good whether you want a relative or an absolute quantification of your qPCR products. The standard curve will show you how efficiently your primers amplify your target, and I think this information is essential to be absolutely sure about the results of your assay at the end.
if the primers that I am using has been reported in conducting the quantitative real time PCR, must I generate the standard curve? is it permitted to use these primers directly in your paper without generating the standard curve, if you want to publish your paper?
Hi Liang
You should always run a standard curve because primers synthesis is not always the same neither is the purification. I like to run the standard curve every time I use a new primer or a new batch. Once I ordered a set of primers from an article and when I checked primer efficiency it was really low. I had to redesign them (which I prefer).
Let me share with you my procedure before qPCR
1. I run a normal PCR to check for primer dimer, single amplicon and I always sequence the amplicon.
2. I run an titration curve using a 3x3 matrix and fixed DNA
3. I run an efficiency curve with the best primer concentration
Hi Liang
Yes it is always better to do std curve.
Sometimes when reviewers are critical they can ask you for the standard curve. Apart from that it will give a better idea about the efficiency of your primers and a rough idea about the expression of your gene of interest.
- Badges
- Science topic