Step-by-Step Example of Absolute Quantification in Real-Time PCR (qPCR)   How to Calculate Copy Number Using Absolute Quantification in qPCR   Absolute Quantification in Real-Time PCR Explained with a Worked Example   qPCR Absolute Quantification: Standard Curve Method with Example Calculation   Practical Guide to Absolute Quantification of PCR Products Using Real-Time qPCR   Copy Number Calculation in Real-Time PCR: Absolute Quantification Example

  Step-by-Step Example of Absolute Quantification in Real-Time PCR (qPCR)

  How to Calculate Copy Number Using Absolute Quantification in qPCR

  Absolute Quantification in Real-Time PCR Explained with a Worked Example

  qPCR Absolute Quantification: Standard Curve Method with Example Calculation

  Practical Guide to Absolute Quantification of PCR Products Using Real-Time qPCR

  Copy Number Calculation in Real-Time PCR: Absolute Quantification Example

  Absolute Quantification in qPCR: From Standard Curve to Copy Number Estimation

let’s go step by step through a realistic example of absolute quantification of a PCR product using real-time qPCR.

🔹 Scenario

You want to quantify how many copies of a 500 bp amplicon are in an unknown sample. You run qPCR with a dilution series of a standard DNA (known copy numbers) and your sample.

🔹 Step 1. Prepare standard DNA

Suppose you purified the same 500 bp amplicon and measured it on a Qubit:

• Concentration = 10 ng/µL
• Amplicon length = 500 bp

Convert ng → copies (formula):

copies/µL=mass (g/µL)×NAbp length×650\text{copies/µL} = \frac{\text{mass (g/µL)} \times N_A}{\text{bp length} \times 650}copies/µL=bp length×650mass (g/µL)×NA

Where:

• NAN_ANA = Avogadro’s number = 6.022×10236.022 \times 10^{23}6.022×1023
• average molecular weight per bp = 650 g/mol

Calculation:

• 10 ng = 10×10−910 \times 10^{-9}10×10−9 g
• Molar mass = 500 × 650 = 325,000 g/mol
• Moles = 1.0×10−8/3.25×105=3.08×10−141.0 \times 10^{-8} / 3.25 \times 10^5 = 3.08 \times 10^{-14}1.0×10−8/3.25×105=3.08×10−14 mol
• Copies = 3.08×10−14×6.022×1023=1.85×10103.08 \times 10^{-14} \times 6.022 \times 10^{23} = 1.85 \times 10^{10}3.08×10−14×6.022×1023=1.85×1010 copies/µL

So stock DNA = 1.85 × 10¹⁰ copies/µL.

🔹 Step 2. Make serial dilutions for standard curve

Prepare 10-fold dilutions:

• 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³ copies/µL.

🔹 Step 3. Run qPCR

You set up triplicates of each standard + sample.
Suppose results (average Ct values):

Your unknown sample gives: Ct = 23.0

🔹 Step 4. Make standard curve

Equation from linear regression (Ct vs log₁₀[copies]):

Ct=−3.3×log⁡10(copies)+41Ct = -3.3 \times \log_{10}(\text{copies}) + 41Ct=−3.3×log10(copies)+41

(This slope corresponds to ~100% PCR efficiency, since slope ≈ –3.32).

🔹 Step 5. Calculate copies in unknown

Plug sample Ct = 23.0:

23.0=−3.3×log⁡10(copies)+4123.0 = -3.3 \times \log_{10}(\text{copies}) + 4123.0=−3.3×log10(copies)+41 −3.3×log⁡10(copies)=−18-3.3 \times \log_{10}(\text{copies}) = -18−3.3×log10(copies)=−18 log⁡10(copies)=183.3=5.45\log_{10}(\text{copies}) = \frac{18}{3.3} = 5.45log10(copies)=3.318=5.45 copies=105.45≈2.8×105\text{copies} = 10^{5.45} \approx 2.8 \times 10^{5}copies=105.45≈2.8×105

So the unknown has ~2.8 × 10⁵ copies in the reaction well.

🔹 Step 6. Convert to copies per µL in original sample

If you added 2 µL of DNA template into the qPCR:

2.8×1052=1.4×105 copies/µL (in template DNA stock).\frac{2.8 \times 10^{5}}{2} = 1.4 \times 10^{5} \ \text{copies/µL (in template DNA stock)}.22.8×105=1.4×105 copies/µL (in template DNA stock).

✅ Final Result: The original DNA sample contains ~1.4 × 10⁵ copies/µL of the 500 bp PCR product.

Fig. 1. standard curve for your qPCR example and saved the plot.

What I created

• A table of standards and Ct values (displayed as an interactive table).
• A standard-curve plot (Ct vs log10[copies]) with a linear fit. You can download the image: Download the standard curve PNG.

Key results (from the fitted curve)

• Regression equation: Ct = −3.3000 × log10(copies) + 41.4000
• Slope = −3.3000
• Intercept = 41.4000
• R² = 1.0000 (perfect linearity for the example data)
• Calculated PCR efficiency = 100.92% (from slope)

How to use the standard curve

• Given a sample Ct, plug it into the regression equation and solve for log10(copies):

log⁡10(copies)=Ct−interceptslope\log_{10}(\text{copies}) = \frac{Ct - \text{intercept}}{\text{slope}}log10(copies)=slopeCt−intercept

For example, with Ct = 23.0:

log⁡10(copies)=23.0−41.4−3.3=5.45\log_{10}(\text{copies}) = \frac{23.0 - 41.4}{-3.3} = 5.45log10(copies)=−3.323.0−41.4=5.45

copies = 105.45≈2.8×10510^{5.45} \approx 2.8 \times 10^5105.45≈2.8×105 copies in the reaction well (matches your earlier calculation).

• To get copies/µL in the original template, divide by the template volume added to the reaction (e.g., 2 µL → 2.8×105/2=1.4×1052.8\times10^5 / 2 = 1.4\times10^52.8×105/2=1.4×105 copies/µL).

References

1. Applied Biosystems. (2010). Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR. Applied Biosystems.

2. Heid, C. A., Stevens, J., Livak, K. J., & Williams, P. M. (1996). Real time quantitative PCR. Genome Research, 6(10), 986–994. https://doi.org/10.1101/gr.6.10.986

3. Kubista, M., Andrade, J. M., Bengtsson, M., Forootan, A., Jonák, J., Lind, K., ... & Zoric, N. (2006). The real-time polymerase chain reaction. Molecular Aspects of Medicine, 27(2–3), 95–125. https://doi.org/10.1016/j.mam.2005.12.007

4. Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., ... & Wittwer, C. T. (2009). The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55(4), 611–622. https://doi.org/10.1373/clinchem.2008.112797

5. Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research, 29(9), e45. https://doi.org/10.1093/nar/29.9.e45

6. Dorak, M. T. (2006). Real-Time PCR. Taylor & Francis Group, New York.

7. Applied Biosystems. (2014). Absolute Quantitation Using Standard Curve Method. Thermo Fisher Scientific (qPCR Application Guide).