The Q Calculator uses the ΔΔCt method to provide a straightforward and effective means for comparing gene expression levels across different samples. Here’s what you need to know about the formula it uses:
ΔΔCt = (Ct target gene – Ct reference gene) treatment – (Ct target gene – Ct reference gene) control
Understanding the Formula
- Ct target gene: This is the threshold cycle at which the fluorescence of the PCR product from the gene of interest is detectable above the background level. It indicates the cycle number at which the sample’s gene expression reaches a significant threshold.
- Ct reference gene: A consistent and stable gene used as a baseline to normalize gene expression levels across different samples.
- Treatment and control samples: Treatment refers to the sample under investigation (possibly treated with some reagent), and control is a baseline or standard sample for comparison.
Assumptions for Accuracy
To ensure accuracy, the Q Calculator assumes that the PCR efficiencies for the target and reference genes are approximately equal. If this isn’t the case, adjustments using methods like the Pfaffl method, which accounts for different efficiencies, might be necessary.
Practical Application: A Step-by-Step Guide
Using the Q Calculator is straightforward:
- Select the appropriate genes: Choose your target and reference genes. The reference gene should exhibit stable expression across all samples.
- Input Ct values: Enter the Ct values from your qPCR for both treatment and control samples.
- Calculate ΔΔCt: Use the provided formula or an online Q Calculator tool to compute the ΔΔCt value.
- Interpret the results: A positive ΔΔCt indicates an increase in gene expression, while a negative value suggests a decrease compared to the control.
General qPCR Terms and Conversions
| Term | Definition | Typical Values or Conversions |
|---|---|---|
| Ct (Cycle Threshold) | Cycle number at which fluorescence exceeds the threshold. | Variable; depends on qPCR efficiency and sample |
| ΔCt (Delta Ct) | Difference in Ct values between the target gene and reference gene. | ΔCt = Ct (target gene) – Ct (reference gene) |
| ΔΔCt (Delta Delta Ct) | Difference in ΔCt values between treated and control samples. | ΔΔCt = ΔCt (treated) – ΔCt (control) |
| Fold Change | Measure of the change in gene expression relative to the control. | 2−ΔΔCt (if ΔΔCt is negative, gene expression is upregulated) |
| Efficiency (E) | Efficiency of the PCR reaction, typically assumed to be close to 100%. | 90%-110%; E = 10−1/slope of the standard curve |
| RQ (Relative Quantity) | Relative quantity of the target gene normalized to the reference gene. | RQ = 2−ΔCt |
| Normalization Factor | Factor used to adjust the Ct values of samples for comparison. | Calculated based on reference gene(s) Ct values |
Example of Q Calculator
Let’s go through an example to clarify the process:
- Sample Preparation: Suppose we are studying gene X under drug treatment.
- Perform qPCR: Obtain Ct values for gene X and a reference gene (e.g., GAPDH) for both treated and untreated cells.
- Calculate ΔΔCt: Input these Ct values into the Q Calculator.
- Result Interpretation: Analyze the output to understand the effect of the drug on gene X expression.
Most Common FAQs
It’s the point where the fluorescence of the PCR product is first detectably above the background, reflective of the gene expression level.
Choosing the right reference gene is crucial for accurate normalization of qPCR data. A reference gene should have stable expression across all samples and conditions in the experiment to serve as a reliable baseline
Amplification in negative controls (no-template controls) suggests contamination or setup errors. To address this:
Re-assess the PCR mix and preparation area for possible sources of contamination.
Prepare new reagents and run the experiment again.
Ensure that all pipettes and surfaces are clean and that the PCR setup is performed in a contamination-free environment.