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.
The table below shows Endpoint PCR cycle numbers for Universal Human Reference RNA (UHRR) input amounts. This should be taken as a guideline only! Optimal cycle numbers could exceed these ranges depending on the sample type (i.e., species, tissue, RNA quality e.g., FFPE RNA, etc.). 
ATTENTION!: These cycle numbers are provided as a reference only and will not suit all sample types!
qPCR Assay for Endpoint PCR Cycle Number Determination
The qPCR assay to determine optimal PCR cycle numbers for library amplification is performed using an aliquot of purified double-stranded cDNA, prior to the final library amplification.
To perform the qPCR assay you will need:
The PCR Add-on Kit for Illumina (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)
ATTENTION: The use of SYBR Green I-containing qPCR mastermixes from other vendors is not recommended.
The recommended protocol for the qPCR assay is provided in the PCR Add-on Kit Instruction Manual, as well as in the Appendices of each QuantSeq 3' mRNA-Seq Library Prep Kit User Guide.
Briefly, the cDNA obtained after second strand cDNA synthesis and purification, is topped up to a total volume of 19 μl using Elution Buffer (EB).
Next, setup a PCR using 1.7 μl of this cDNA is used as template plus:
7 μl PCR Mix (PCR)
1 μl Enzyme Mix (E)
5 μl P7 primer (7000), and
1.2 μl of 2.5x SYBR Green I dye (final concentration must be 0.1x)
Make up the total volume to 30 μl using Elution Buffer (EB) or molecular biology-grade water.
IMPORTANT! The final concentration of SYBR Green I dye must not exceed 0.1x! Higher concentrations can inhibit amplification.
The qPCR is performed in a real-time PCR machine for at least 35 cycles.
Calculating the Endpoint PCR Cycle Number
The amplification curves from the qPCR (set to linear scale) are then used to determine the cycle number corresponding to 50 % of the maximum fluorescence (50 % MF cycle No.) reached at the amplification plateau (See figure below).
The final number of Endpoint cycles to use is then calculated by the formula:
No. Endpoint PCR Cycles = 50 % MF cycle No. - 3 cycles
The reason 3 cycles are subtracted from the 50 % MF cycle number, is becuase 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. 