These These guidelines are relevant for calculating endpoint PCR cycle numbers for QuantSeq V2 with UDIs, QuantSeq-Pool and CORALL RNA-seq Seq V2 library prep protocols. Please also see the respective User Guides for kit-specific details.
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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.
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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.
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Figure: Example of Endpoint PCR cycle number calculation from qPCR amplification curve (linear scale).