Using Standard Curve to Estimate DNA Quantity - Forensic Focus #4

Submit your questions at http://www.thermofisher.com/forensicf...

Everyone wants to know how much DNA is in their extract, but then they ask: how can I tell if my estimate is accurate?

The standard curve holds the answers.

A standard curve is a tool that allows us to estimate the DNA concentration of unknown samples by comparing them to standards with known DNA concentrations.

In this example, the standards consist of a 10-fold dilution series ranging from 50 ng/ul down to 5 pg/ul.

During each PCR cycle, the amount of fluorescent signal for each standard in the dilution seies is measured. When the fluorescent signal crosses the detection threshold the cycle number is recorded as a Ct value, or threshold cycle value. The Ct value is what ultimately is used to create the standard curve.

The Ct values are inversely proportional to the concentration of DNA in the standards. The high-concentration, 50 ng/ul standard will cross the detection threshold first, generating a “low” Ct. The low-concentration, 5 pg/ul standard will take many more cycles to cross the same threshold - and therefore the Ct will be higher.

The Ct values for each dilution of the standard curve are plotted on a graph, and the software generates a regression line that fits the data.

Because the standards are 10-fold dilutions, we expect the change in Ct from one standard to the next to be uniform. An uneven distribution of Ct values might indicate that the dilution series was not accurately pipetted.


Let’s take a look at the standard curve for a specific DNA target, the small autosomal target.

The X axis is the log of the known standard concentrations. The Y axis is the Ct value of each standard.

Now, Do you see the quality metrics at the bottom of the screen? Let’s review Slope, Y intercept, and R2.?

The slope measures the efficiency of the PCR reaction. In a perfect world, a slope of -3.3 indicates that the PCR reaction is 100% efficient; the target DNA is doubled each cycle. Two copies become four; four become eight; and so on.

The Y intercept is the expected Ct value for a 1ng/ul sample.

The R2 value measures how well the regression line fits the data points. A line that fits the data points perfectly has an R2 of 1. If your data points are scattered, the R2 value for the line will be lower.

The Ct values of your standards affect the slope, the Y intercept, and the R2 value. It is very important to prepare the standard dilution series carefully to ensure consistent and accurate results!

Running the standards in duplicate can help ensure you have a high quality standard curve.

Once your standard curve passes the metrics test, it can be used to evaluate an unknown sample!

The Ct value of the unknown sample is measured, and compared to the standard curve to estimate the DNA concentration of the unknown sample. Couldn’t be simpler!

That’s it for today. If you have other questions, just click on the link below.

And don’t forget- when in doubt, refer Back to the Bases!