Lexogen Online FAQs

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).

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