Hello everyone, I am very new to q PCR and I am running a Taq Man probe-based q PCR assay to detect Toxoplasma gondii DNA in tissue samples of animals. I have generated a standard curve using 8 serial dilutions (starting from 500pg/ul to 0.00005pg/ul) using T. gondii g Blocks (synthetic DNA). My product size is 81bp.
At the highest dilution (0.00005pg/ul) I received a mean Ct value of 31.49. Do I need to use few more dilutions to get a higher Ct value? Is there any recommended reliable range of Ct values? The Ct value I am gaining from my standard curve will be used to determine whether the tissue sample is positive or negative for T. gondii. Any comments on the standard curve are also appreciated. Would love to hear the opinion of others who are doing q PCR.!
I recommend that you do further dilutions to identify the reliable highest dilution that gives you an acceptable sigmoidal curve. Also, if this setup is non-proprietary (i.e., not from a commercial kit), then I also suggest that you determine its analytical parameters (e.g., limit of blank, limit of detection, limit of quantification).
In my experience, if the intended maximum number of cycles is 45.00, a sensitive threshold for positivity is set at 40.00 in practice. However, there is arguably some arbitrariness on this, and I would rather that you correlate the threshold of positivity with some relevant metric (e.g., if you have the resources to culture T. gondii, you can determine the highest dilution of the protozoan that can demonstrate culture growth within a specified period of time in coordination with your institutional expert on protozoan/microbial culture - then determine the reliable CT reading for this dilution in qPCR).
The following references may help:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5496743/
https://www.cell.com/trends/biotechnology/fulltext/S0167-7799(18)30342-1
Rule of thumb is "35 cycles is enough to amplify even a single template to detection" (i.e. Cq ~35 = 1 initial molecule).
With efficiency differences this can be 34-38 in practice, but as a general QC check anything in the 30s should be considered to be 'countable numbers of transcripts'. You can already see that your most diluted sample is variable: this is exactly what is expected when you get into the stochastic range.
What does this mean for your assay? Well, it really depends on what you expect to see. I would imagine most infections would be either "yes" or "no", with either loads of signal, or barely any (or none). If this is the case, it doesn't matter as much where you put the cut-off, because you'll have a big range to play with. If you expect there to be more "low level", edge case scenarios, then it's worth establishing the lower limit of detection, as suggested by Aedrian Abrilla .
So yeah, you could go lower on your standard curve.
Aedrian Abrilla It would be a good idea, I'll run it with some more further dilutions and see how it's going. Same time it would be good to correlate the cut-off with another relevant metric. Thank you very much.!
John Hildyard Thank you very much. That helped a lot. I should note down the rule of thumb, didn't know that before. Great.!
How long is your template? It is very likely that you have less than one molecule of your template per ul in the lower concentration samples. It may help to calculate how much template you have to add to have one molecule and call that Ct as your limit of detection (I would use more than one molecule per reaction - reliability at one molecule probably will be so so).
Aidas Nasevicius Hi thank you for your answer. My template is 81bp. How can I use that value to detect what you have mentioned? It would be great to have that calculation.!
Tharaka Liyanage Both product and template are 81bp long?
If we assume that average molar mass of a DNA basepair is 1000g/mol, then 81bp dsDNA molar mass is 81000g/mol. 0.00005pg (=5*10^-17g) of your template then would be 6.17*106-22 mol.
1 mol = 6.022*10^23 molecules. Therefore, 6.17*10^-22 mol of your dsDNA template is equal to about 370 molecules.
I hope this makes sense...
Tharaka Liyanage Ah, then at your lowest concentration, 1 ul will have about 212 copies of the template. So, you probably can do one more 10x dilution.
Aidas Nasevicius I have done three more 10X dilutions in my standard curve (so totally 11 dilutions). I found that the 10th dilution is reliable, in the 11th dilution qPCR doesn't give a reliable amplification. The concentration of my 10th dilution is 0.000 0005 pg/ul.
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