Do we need to adjust threshold to certain value or select specific one and use it throughout the experiments? Such as if we compare two samples treated/untreated we set same threshold (will it be automatically set the same)?
Some researchers in our lab "just use their 0.1" when they do analysis.
Setting threshold has often being discussed. Automatic one is easy to use but can lead to bad quantification if the curves are not well sigmoid shape. As for me working for years on quantification of environment matrices, it is good to have a reference to set up your threshold. If you work with standard curve, set up your threshold with them and adapt slightly considering the shapes of sample curves and the exponential phase of the sigmoid. If not using standards, try to use a gold standard, one calibrated target DNA supposed to amplify always the same way and giving the same Ct. Il will permit you to set up correctly your threshold, because I would say that any run is different even with very good performance of any PCR supplier.
Automatic threshold is good when curves are ideal, less if not. Be always critical to your results..
Good luck...
Best way to rule out possible variability between different qPCR experiments is to use a day to day control. Ideally a cell line cDNA that could also be used as a positive control for other target genes. You simply include this sample in each plate and set the treshold line so that you get identical CT values after each run.
Setting threshold has often being discussed. Automatic one is easy to use but can lead to bad quantification if the curves are not well sigmoid shape. As for me working for years on quantification of environment matrices, it is good to have a reference to set up your threshold. If you work with standard curve, set up your threshold with them and adapt slightly considering the shapes of sample curves and the exponential phase of the sigmoid. If not using standards, try to use a gold standard, one calibrated target DNA supposed to amplify always the same way and giving the same Ct. Il will permit you to set up correctly your threshold, because I would say that any run is different even with very good performance of any PCR supplier.
Automatic threshold is good when curves are ideal, less if not. Be always critical to your results..
Good luck...
May I call HKG a gold standard? I did few standard curves for it and know how it behaves under various concentration and what threshold to expect.
Then...how do I adapt my thresholds? If one day my HKG gives me lower/higher value then usual, I adapt it till "usual" numbers and in parallel increase/decrease threshold for other genes? Say, lower threshold by 0.05 to all genes. Is that what you mean?
From my understanding, If I compare control group 1 with treated group 2, for the same GOI1 threshold should be the same for these two groups? Say, if they carry different names GOI1_control and GOI1_treated then software may assign separate thresholds for the same gene. Is it correct? As I noticed when I give to my GOI same name (without defining condition 1 and 2): just GOI1 under "Unknown task", it gives same threshold for both conditions, but shows error "high St deviation". [As Ct for my control group and treated apart by 5 cycles]
If you are not using the Cy0 Method (http://www.cy0method.org/) then you need to be sure to use the exact same threshold on a target basis. E.g. GOI one uses 1 and only one threshold and reference gene (HKG) uses 1 and only 1 threshold from plate to plate. If you use an inter-plate calibrator for each different GOI and reference gene, then you would have the rationale to use a different threshold for each target and ref. gene on each different plate. The beauty of the Cy0 Method is that is never assumes the same threshold line across all amplification curves. It uses Richards 5-parameter sigmoid modeling to determine the true tangent to each amplification, and then draws a line to the x axis to give Cq values that are then indicative of the relative target differences between samples.
If you always use the same mastermix, and prepare your samples identically, and set up the mastermix carefully the same way each time, and the machine is in excellent working order, the threshold should not change for a certain target. E.g. For 14 targets I've studied for the past 8 years in sheep lung, I have used a characteristically different and unique threshold for each different target - and from experiment to experiment, those threshold values have all been good. Quantification itself is affected if using a different threshold (per target) per sample and/or per plate for a certain target -- unless you are using the Cy0 Method.
- Badges
- Science topic