Dear all
I was trying to determine T-cell receptor (TCR) variant by sequencing from qRT-PCR product.
Of qRT-PCR, I had optimised template concentration, annealing temp. and gave result with ΔCt of 4–8 between sample of experiment and NTC. There was positive signal of NTC after cycle 30th (out of 35–40 cycles performed.).
Of sequencing, I did gel visualisation instead of performing melt curve for determining primer specification and reaction condition (whether or not unspecific band appeared.). Ahead of cycle sequencing, gel extraction performed to minimise reaction residues. The result somehow slightly confusing. In DJ-region, which was the sequence of interest, had more than one sequence. Interestingly, sequencing result of V- and C-region (flanking DJ-region) yielded clear peaks, as expected.
What issues that might take into account?
I would be very happy and appreciate for every suggestion.
Regards.
PS. I skipped melt curve analysis to avoid denaturation of the template.
You may have PCR products of similar sizes within one band on a gel; clone and sequence your PCR products. Which cell type(s) are you analyzing?
A delta Cq of 4-8 indicates a not well optimized RT-qPCR. I presume you use a DNA binding dye?
In a well designed PCR NTC's if any, should dffer at least 10 Cq from your experimental sample. To much primers or badly constructed primers will give a low Tm melting peak.
When we optimize qPCR we perform a gradient PCR with 50 cycles. Then every possible artifact will appear. At best annealing temperature we titrate the primers in a checkerboard going from 0,031 up to 0,4 uM, we measure Cq, delta Rn, look at the quality of the amplifiction curve (is it really log-lenei for at least 9 cycles?) and perform a melting curve. First use primer pairs e.e. 0,031/0,031 op to 0,4/0,4. At the best combinations re tested in a checkerboard, analysed once more. The best of them re subjected to garose electrophoresis. We often use REV/FW primers at different concentrations
To sequence quite comportable, you amplicon should be above 120 NT
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