I've seen in MIQE guidelines that calibration curves a needed when performing RT-qPCR. I'll use genomic DNA to perform these calibration curves.
My question is that I don't know the range of concentrations to test.
The genes I will check for differential expression have logFC between 2-8 (base 2 log).
50 ng/μL is the sample concentration I will have when performing the Comparative CT Experiment, but 98% is ribosomal RNA.
Please let me know your suggestions and possible reference books/papers to revise.
To begin with, you must have an idea of what sort of CT values to expect from your test samples. If you don't know, easy to do a few test runs and find out. Then prepare dilution series to cover this range. If it varies a lot or is unpredictable, prepare dilution series for full range, ie; from approximately CT 10 to CT 34.
To do this, make a high concentration and serially dilute it 10-fold to make about 12 dilutions. Do a test qPCR on your dilutions and pick out about 8 or minimum 5 that worked and covers a good range. Remember to do your test runs in triplicates so you get a nice error range.
Hi Roy,
Thanks for your quick response, I really appreciate you help. Before reading your answer I arrived to a similar conclusion but its great to agree with you.
Since I don't know CT values of my samples, I would have to do a few test runs as you suggest. Should I test all my targets? Right?
Yes......you should check all the targets across all dilution because expression of each gene vary from another. Select the dilution limit in which even low expressing target have Ct value less than 35.
If you want to apply comparative Ct value method for analysis, paper from schmittgen (NATURE PROTOCOLS , VOL.3 NO.6 , 2008) will be helpful.
- Badges
- Science topic