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# qPCR Efficiency Calculator

What is qPCR efficiency?How to calculate qPCR efficiency — FormulaHow to use the qPCR efficiency calculator — ExampleHow to optimize PCR efficiency and dilution curvesFAQs

Use this qPCR efficiency calculator to determine the efficiency of your PCR assay from the slope of the standard curve. To verify that your PCR runs efficiently, you should get an acceptable qPCR efficiency of 90-100%, meaning that the number of molecules in the target sequence doubled during each replication cycle.

Keep reading to find out how to calculate primer efficiency, how to interpret the qPCR efficiency results correctly, and find tips on how you can improve your PCR results. You will also find an example table of a plate layout for DNA dilution to reach better amplification rates 🧬. If the terminology gets you confused already, it might be best to look at our annealing temperature calculator first to understand the fundamentals of PCR better.

## What is qPCR efficiency?

PCR efficiency, expressed in percentage (%), tells us how well our PCR (polymerase chain reaction) assay performed. It looks at the doubling ratio of the target sequence. If the qPCR (quantitative PCR) primer efficiency equals 100%, it means that our target sequence doubled in each PCR cycle and the polymerase enzyme and primers are working at maximum capacity; that is our goal.

We calculate the PCR efficiency to determine our assay's reliability and accuracy and to compare different assays against each other. Normally, a standard curve experiment with a dilution series is run. The resulting Ct (cycle threshold) value is plotted against the log of the nucleic acid input level to generate a linear graph. We use the slope or gradient of this graph to determine the PCR reaction efficiency (if you need a refresher on that topic, take a look at our slope calculator).

An efficiency of <90% or ≥110% is unacceptable and indicates that further assay optimization is required. Let's look at the use of our qPCR efficiency calculator, the equation, and how to interpret the results correctly next! 👩🏻‍🔬

🙋 What was the Ct value again? It is the number of PCR cycles required for the fluorescent signal to exceed background levels. Ct levels are inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Ct level, the greater the amount of target nucleic acid in the sample).

## How to calculate qPCR efficiency — Formula

To calculate the PCR efficiency, we need to know only one value: The slope of the standard curve. To get this value, run a dilution curve and look at the slope of that resulting curve. At this point, you can approximate the PCR efficiency: if the slope has a value of ≈-3.3, your PCR runs with approximately 100% efficiency, and a slope of -3.322 equals precisely 100% (-1/log(2)). This means that the Ct values of a 10-fold dilution will be 3.322 cycles apart. If the value is lower, e.g., -3.8, it indicates that your PCR needs to be improved.

To determine the exact value of your PCR efficiency, we use the following qPCR efficiency formula:

$\text{Efficiency} = -1 + 10^{(-1/\text{Slope})}$

The result of this equation is the efficiency in percentage. If you reach a value of 100% — perfect, your PCR is running with total efficiency. Remember that only values between 90% and 110% are acceptable!

Your PCR assay must still be improved if you reach a lower value. You might also calculate values over 100%; what happened here? Even though a value above 100% is practically not possible, this value can theoretically be calculated because of many factors such as contamination, dilution point mixing problems interfering with the PCR, or when values are in the non-linear phase of the reaction.

The weakest point in determining PCR efficiency is the preparation of the dilution curves, which causes incorrect efficiency calculations. Our serial dilution calculator is only one of Omni Calcuator's dilution calculators that can help you make correct solutions. Also, take a look at the section "How to optimize PCR efficiency and dilution curves" below to find tips and tricks on how to improve the accuracy of the dilution curve and PCR performance as well as an example table of a plate layout for DNA dilution. 🧪

## How to use the qPCR efficiency calculator — Example

The qPCR efficiency calculator assumes that on your standard curve diagram, the log of DNA copies is plotted on the x-axis, and the Ct is on the y-axis. First, verify that your diagram looks like this! After, you can follow these easy steps:

1. Find out the slope of your standard dilution curve; let's take a value of -3.35 as an example.

2. Insert the slope value into the first field of the calculator.

3. That's all you need to do! The calculator will determine the qPCR primer efficiency in percent, as well as the amplification factor as follows:

• Efficiency = -1 + 10(-1/-3.35) = 98.84%; and

• Amplification factor = 10(-1/-3.35) = 1.99.

The amplification factor can have a maximum value of 2 because DNA has two strands, and at maximum, a target sequence can double each cycle.

You can also calculate the other way around to get the value of the slope from the primer efficiency:

• -1/log1.9884 = -3.35
4. Interpret the results: If your efficiency lies between 90% and 110%, you can happily accept it. But what if the result lies out of these acceptable qPCR efficiency bounds? Let's look at what we can do next in this case!

🙋 Sample preparation, pipetting dilutions, and waiting for the robots to finish can be monotonous tasks with long waiting periods. Our podcast calculator can help you spend this time more efficiently. 😉

## How to optimize PCR efficiency and dilution curves

Find some additional suggestions here on how you can improve your standard curve and PCR efficiency! We will also give you a template for piping the DNA dilutions on your well plate for the standard curve PCR run. In the first place, make sure you follow the provided guidelines for the PCR kits and reactants you use! 🔬

• Use at least 5 dilution points with 10-fold dilutions;

• Run at least triplicates of each dilution point;

• If you have 1 or more outliers in the standard curve, repeat the curve;

• Check R² and outliers after the run;

• Use the four eyes principle — let more than one person perform the standard curve for the first time;

• Use only pure template DNA (avoid contamination) and keep your workspace clean and disinfected (separate workspace for different tasks) 🦠;

• Make sure your pipetts are properly calibrated and your material is appropriate and standard material for the assay you want to perform;

• Verify the specificity with fluorescent DNA-binding dyes.

There are various methods to verify the mentioned points. Always investigate abnormal outcomes carefully and consider different reasons for fault. Try to change only one or a few parameters at a time to determine precisely what caused the error initially. Below, you will find a template for the DNA dilutions on your well plate for the standard curve:

Plate / Column

1

2

3

4

5

6

Rest of plate

A

1

1

1

1

B

0.1

0.1

0.1

0.5

0.5

0.5

C

0.01

0.01

0.01

0.25

0.25

0.25

D

0.001

0.001

0.001

0.125

0.125

0.125

E

0.0001

0.0001

0.0001

0.0625

0.0625

0.0625

F

0.00001

0.00001

0.00001

0.03125

0.03125

0.03125

G

0.000001

0.000001

0.000001

0.015625

0.015625

0.015625

H

NTC

NTC

NTC

NTC

NTC

NTC

💡 NTC stands for no template control or negative control and is usually sterile water used to detect potential contamination or non-specific amplification in the qPCR assay.

FAQs

### How do you calculate amplification efficiency for qPCR?

To calculate the PCR efficiency in percentage (%):

1. Run a standard curve PCR with dilutions and note the resulting curves slope, e. g., -3.35.

2. Take the qPCR efficiency formula and insert your slope value:

• Efficiency = -1 + 10(-1/-3.35) = 98.84%
3. You can also calculate the amplification factor:

• Amplification factor = 10(-1/-3.35) = 1.99
4. Interpret your result: A slope of exactly 3.322 means 100% PCR efficiency, while results between 90% and 110% are acceptable. Higher or lower results indicate that the PCR assay needs further improvements to perform efficiently.

### How to improve qPCR efficiency?

Some of the most critical parameters to improve qPCR efficiency results are:

• For the standard curve, use ≥ 5 dilution points and run at least triplicates for each;

• Repeat the assay if you have outliers;

• Use the 4 eyes principle on all process steps;

• Use only uncontaminated template DNA and keep a clean workspace;

• Use correctly calibrated pipettes;