These guidelines are relevant for calculating endpoint PCR cycle numbers for QuantSeq V2 with UDIs, QuantSeq-Pool, and CORALL RNA-Seq V2 library prep protocols. Please also see consult the respective User Guides for kit-specific details.
The final number of Endpoint cycles to use is calculated by the following formula:
No.Endpoint PCR cycles = 50% MF cycle No – 3
50% MF cycle No = Cycle number corresponding to 50% of the maximum fluorescence value that is reached at the amplification plateau.
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 settingSet 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).