These following These guidelines are relevant for calculating the correct number of cycles to use for endpoint PCR cycle numbers for with the following kits: QuantSeq V2 with UDIs, QuantSeq-Pool, and CORALL RNA-Seq V2library prep protocols. Please consult 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.
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!
3 cycles are subtracted from the 50 % MF cycle number because the input used for Endpoint PCR (17 μl of cDNA) is 10X the input used for the qPCR assay (1.7 μl of cDNA). This corresponds to a difference in ~ 3 PCR cycles.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 bebeing overcycled. overcycling.
Tips for calculating Endpoint PCR cycle numbers correctly:
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