Hi Yara,
It is the dynamic range over which a reaction is linear (R²≥0.98), from the highest to the lowest quantifiable copy number established by means of a calibration curve. For qPCR experiment results, the only valid Cq values are those that fall into the validated dynamic range. It is recommended to use template concentrations within the linear dynamic range for qPCR, which results in a Cq of between 20 to 30. Usually, a 10-fold dilution series is sufficient to cover the most logs of dynamic range.
I recommend this paper by Stephen A Bustin et al., The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, Clinical Chemistry, Volume 55, Issue 4, 1 April 2009, Pages 611–622, Article The MIQE Guidelines: minimum Information for Publication of ...
Best regards,
Clei
Dynamic range and sensitivity are like synonyms. What I understood is the signal in form of the C-value you get at the lowest concentration of the sample till a higher range in the log scale is your dynamic range. For example, a six-order magnitude of dynamic range is better than a four-order magnitude.
1. Enhanced dynamic range is advantageous for the accuracy and precision of experiments.
2. The dynamic range is determined by the total number of positive partitions in the reaction. Which is a confirmation that we will get signals from each partition. The more the number of partitions, more is the more dynamic range.
3. Minimum sample(cDNA of interest) input volume gives you sensitivity(C-value). YOu can use it to plot graphs against volume input and put it into a straight line until you get the linear dynamic range out of each volume. For subsequent dilution.
The maximum volume level a system tolerates will give you the upper limit of dynamic range and vice-versa for the lowest volume. Together they determine dynamic range.
Source: https://doi.org/10.1016%2Fj.bdq.2016.10.001
Qiagen.com
For qPCR, it means being able to make accurate measurements (R2 is ≥0.980) around certain range of concentrations be it starting copy numbers or input DNA/RNA. I.e. a diagnostic RNA assay might want to be able to detect gene fusions as little as 1 copy per reaction up to 100,000 copies. In this scenario we would need 6 orders of magnitude (see below) to be able to have confidence that we can detect 1 or 100,000 copies (accurately).
1) 10^0 copy (1 copy, arguably sometimes under certain conditions)
2) 10^1 copies (10 copies)
3) 10^2 copies (100 copies)
4) 10^3 copies (1000 copies)
5) 10^4 copies (10,000 copies)
6) 10^5 copies (100,000 copies)
6-8) 10^6-8 copies (1,000,000 copies and onwards)
You generally (sometimes) have problems at the low & very high copy numbers and between very low & very high DNA/RNA inputs (see figure 3-https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6252.pdf). You typically find the linear dynamic range by using materials that you know have certain copies or nano/micrograms. PCR is a doubling process. If we start with say 200 copies in a 5 uL sample and dilute that with 5 uL, and rinse and repeat half dilution to 100, 50, and 25 and 12.5 copies, we expect a Ct difference of 1 (because PCR is a doubling process) between 100vs50vs25vs12.5. An easy test for you, take sample you have in the lab that you know the Ct of, dilute it to 1:2 of the original concentration and see what Ct you would get. I.e. if you had a 1:20 cDNA, make a 1:40 cDNA and you will see that your Ct would be delayed by roughly 1 Ct assuming your efficiency is >%90. It is good to know this behaviour, for example digital PCR systems and assays sometimes have problems at the high copy number end.
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