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The optimal number of PCR cycles for a given input amount of total RNA can vary by up to four cycles, depending on sample quality and origin. The optimal cycle number for your specific sample type should be determined using the qPCR assay (see below, and Appendix D).
The table below is provided as a reference only! Optimal cycle numbers could exceed these ranges depending on the sample type (i.e., species, tissue, RNA quality e.g., FFPE RNA, etc.).

The qPCR assay to determine optimal PCR cycle numbers for library amplification is performed using an aliquot of purified double-stranded cDNA produced at step 24. The PCR Add-on Kit (Cat. No. 020.96), and SYBR Green I nucleic acid stain (10,000X in DMSO, not provided in the kit. We recommend using Sigma-Aldrich, Cat. No. S9430 or ThermoFisher, Cat. No. S7585) are required for this qPCR assay.
ATTENTION: The use of SYBR Green I-containing qPCR mastermixes from other vendors is not recommended.
The recommended protocol for the qPCR assay is outlined in Appendix D of the QuantSeq 3' mRNA-Seq Library Prep Kit User Guide.
Briefly, the cDNA obtained at step 24 is diluted with Elution Buffer (EB) to a total volume of 19 μl, and 1.7 μl of this cDNA is then used as template for a qPCR assay containing PCR Mix (PCR), Enzyme Mix 3 (E3), P7 primer (7000), and SYBR Green I dye at a final concentration of 0.1x (NOTE: Higher concentrations of SYBR Green I can inhibit amplification). PCR is performed in a real-time PCR machine for 35 cycles and the amplification curves (in linear scale) are then used to determine the cycle number corresponding to 50 % of the maximum fluorescence reached at the amplification plateau (See Fig. 3, below). Given that 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, 3 cycles are subtracted from this number to give the optimal endpoint PCR cycle number.