I have been trying to optimize gene expression qPCR assays that is already setup in my lab for my genes. However, when I do the PCRs, I am getting late Ct values than expected and thus my standard curve suffers from non-linearity and low efficiency. Also I see that my Ct values are increasing for the same set of primers/ same dilution series of positive control day-by-day.
Things I have tried:
- Use fresh reagents and plastic ware - primers, SYBR, positive control (human reference RNA converted to cDNA), DEPC treated water, filter tips, vials, fumigate working area/ lab.
- Recent calibration of qPCR instrument.
- Reagents have minimal freeze-thaw cycles.
Observations:
- Single melting temp peak, mostly.
- R2 of >0.9 during standard curve generation.
- No NTC contamination.
- Cts for lower dilutions (10, 5 and 1 ng/ul) are usually as expected. Mostly dilutions below 0.5 ng/ul are very late. And thus lower slope (< -4.0) and less efficiency (50-60%) in standard curve generation.
TIA,
Nikhil
It's normal to have an upper and a lower detection limit in your standard curve. Unless you are expecting very, very low expression levels in your experimental samples, you can remove the lowest concentrations from your standard curve. As long as all of the Ct values from the experimental fit on the linear part of the standards, then it OK.
Most folks aliquot out their standards and controls into "single experiment size aliquots" to avoid issues of degradation from repeated freezing & thawing.
Good luck!
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