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 consult the respective User Guides for kit-specific details.
The final number of Endpoint PCR cycles to use is calculated by the following formula:
No. Endpoint PCR cycles = 50% MF cycle No. – 3
The 50% MF cycle No. = value refers to the qPCR cycle number corresponding to 50% of the maximum fluorescence value that is reached at the amplification plateau. at which the 50 % of the maximum fluorescence observed at the amplification plateau is reached (see figure below).
ATTENTION! This value must be calculated manually through analysis of the qPCR amplification curves! The Ct threshold value computed by most qPCR analysis software should NOT be used as the 50 % MF cycle No. Using the automatic Ct threshold may result in under or overcycled libraries!
The reason 3 cycles are subtracted from the 50 % MF cycle number is because the remaining volume of cDNA for endpoint PCR in above mentioned library prep protocols 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 being overcycled.
Tips for calculating Endpoint PCR cycle numbers correctly:
Set the y-axis of the amplification plot to linear scale.
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).