qPCR RNA Quantification Calculator

Standard Curve Equation:

\[ \text{Quantity} = 10^{\left(\frac{\text{Ct} - \text{Intercept}}{\text{Slope}}\right)} \]

RNA Quantity:

Unit Converter ▲

Unit Converter ▼

From:	To:

1. What is qPCR RNA Quantification?

qPCR RNA quantification is a method to determine the amount of RNA in a sample using quantitative polymerase chain reaction (qPCR). The method relies on the relationship between the cycle threshold (Ct) and the initial RNA quantity, established through a standard curve.

2. How Does the Calculator Work?

The calculator uses the standard curve equation:

\[ \text{Quantity} = 10^{\left(\frac{\text{Ct} - \text{Intercept}}{\text{Slope}}\right)} \]

Where:

\( Ct \) — Cycle threshold value from qPCR
\( Slope \) — Slope of the standard curve (typically around -3.32 for 100% efficiency)
\( Intercept \) — Y-intercept of the standard curve

Explanation: The equation converts Ct values to absolute quantities based on your standard curve parameters. The slope represents PCR efficiency, with -3.32 indicating 100% efficiency.

3. Importance of RNA Quantification

Details: Accurate RNA quantification is essential for gene expression studies, viral load measurements, and various molecular biology applications where precise RNA amounts are critical.

4. Using the Calculator

Tips: Enter the Ct value from your qPCR experiment along with the slope and intercept values from your standard curve. The calculator will estimate the RNA quantity in nanograms.

5. Frequently Asked Questions (FAQ)

Q1: What is a typical slope value?
A: For 100% PCR efficiency, the slope is -3.32. Values between -3.1 and -3.6 are generally acceptable.

Q2: How do I obtain the standard curve parameters?
A: Run a dilution series of known RNA quantities, plot Ct vs log10(quantity), and perform linear regression to get slope and intercept.

Q3: What if my PCR efficiency isn't 100%?
A: Use the actual slope from your standard curve. Lower efficiency (flatter slope) will affect quantification accuracy.

Q4: Can I use this for absolute quantification?
A: Yes, this method provides absolute quantification when using appropriate standards of known concentration.

Q5: How should I handle technical replicates?
A: Calculate quantities for each replicate separately, then average the results for more reliable quantification.