I'm trying to detecte very small amounts of a mutant-type mtDNA using SYBR green qPCR. I can't really use a higher concentration of gDNA because my wild-type and housekeeping genes already have a low ct around 17. My negative controls show some primer dimers and are around a ct of 33. However, some samples show a specific melting curve peak identical to the one in my positive control, but with a Ct higher than my negative control (33,34,35) while some samples with the same Ct show a melting curve similar to the negative control, so all over the place or at the primer dimers temperature.
Should I consider aller samples with a higher Ct than my negative control as an absence of the target sequence or should I evaluate each melting curve separately and those showing a specific amplification peak are considered as containing the mutant-type, while others with not showing this peak are considered as homplasmic? If not, is there another way to rigorously discriminate my samples with absence/presence of my target gene?
I mean, how much are the numbers really helping you here?
"Almost always either none, or below the level of quantification" is vaguely useful in the sense that it emphatically communicates "this target is extremely low abundance", but you can't actually put numbers on such low target levels. It's too low to quantify.
It's a PCR, though: it makes a product, and you can run that product on a gel. (this is effectively what the melt curve is pretending to do, really)
If you want to confirm that
A) it's definitely a real product, and
B) it's definitely your GOI
then take those positive and "primer dimer" samples and run them on a gel. If you see sharp single bands in the samples you deem to be positive, and diffuse primer dimer smears in the others, then: primer dimers vs specific amplicon confirmed.
If you then cut out the specific band and send it off for sequencing (cheapo sanger sequencing, using one of your primers) and it comes back with sequence that matches the expected amplicon/target, then "GOI successfully detected" is also confirmed.
Once you have that, you more or less have evidence that you can detect your target if it's there, but also, that even when present, it is present at vanishingly low levels.
I would sort of slightly question the chosen approach, though: if your housekeeping genes (which I assume are either genomic markers or other mtDNA markers) are giving Cqs of ~17, that's ~16-18 cycles earlier than your target of interest, i.e. ~60,000-240,000 greater abundance. That's...a lot, and by extension this implies that your target, even when present, is unlikely to be relevant.
What is your desired outcome measure for this experiment?
I agree with John above. One additional suggestion.... I work with a low expression gene (Cq 30-35) and the validated housekeeping gene has a much lower Cq (19-20). We only dilute the cDNA used in the housekeeping control wells at 1:30. That increases the control Cq about 5 cycles allowing use of more cDNA in all wells (reducing the GOI Cq). As long as you do the same thing for ALL the wells with the housekeeping primer, it does not change your analysis at all.
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