How do I calculate the Endpoint PCR cycle number from the qPCR assay?

These guidelines are relevant for calculating endpoint PCR cycle numbers for QuantSeq FWD/REV, and Flex, and CORALL Total / mRNA-Seq library prep protocols. Please also see the respective User Guides for kit-specific details.

The amplification curves from the qPCR are used to determine the cycle number corresponding to 50 % of the maximum fluorescence that is reached at the amplification plateau (See figure below). This value, we refer to as the 50 % MF cycle No. This value must be calculated manually.

ATTENTION! The Ct threshold value calculated automatically by most qPCR software programs should not be used as the 50 % MF cycle No. Using the automatic Ct threshold may result in under or overcycled libraries!

The final number of Endpoint cycles to use is then calculated by the formula:

No. Endpoint PCR Cycles = 50 % MF cycle No. - 3

The reason 3 cycles are subtracted from the 50 % MF cycle number, is because the remaining volume of cDNA for endpoint PCR is ~17 μl, which is 10x more than the 1.7 μl used for the qPCR assay. In qPCR, 3 cycles are equivalent to a 10x difference in yield.

NOTE: It is usually safe to add one additional cycle to the calculated No. Endpoint PCR cycles without risking that your libraries will be overcycled.

Tips for Calculating Endpoint PCR Cycle Numbers Correctly.

We recommend setting the y-axis of the amplification plot to linear scale. To enable the cycle number to be correctly calculated.
Ensure ROX background fluorescence is turned off.
Draw a horizontal line from the amplification plateau to the y axis and record the value as the Maximum fluorescence value.
Calculate half of this value to get the 50 % MF value.
Draw a horizontal line from this value on the y-axis to intersect the amplification curve, then draw a vertical line from the intersection point to the x-axis.
The cycle number that this vertical line intersects is the 50 % MF cycle No. Round up to the next whole number.
Subtract 3 cycles from this number to get the final Endpoint PCR cycle number.

Figure: Example of Endpoint PCR cycle number calculation from qPCR amplification curve (linear scale).