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The qPCR assay facilitates calculation of the optimal number of Endpoint PCR cycles to use for final library amplification. The assay described below (using the PCR Add-on and Reamplification Kit; Cat. No. 208) is applicable for use with the following library preparation methods:
ATTENTION! The guidelines outlined below do not apply for QuantSeq-Pool or LUTHOR 3' mRNA-Seq library preparation protocols.
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All CORALL RNA-Seq V2 with UDI library prep kits (Cat. No. 171-186, 219-22, 233-234),
All QuantSeq V2 with UDI library library prep kit (Cat. No. 191 – 196, 222-223, 225),
QuantSeq-Pool (Cat. No. 139),
LUTHOR HD (Cat. No. 204, 221),
LUTHOR HD Pool (Cat. No. 205).
Additional Reagents Required
The qPCR assay to determine optimal PCR cycle numbers for library amplification is performed using an aliquot of purified double-stranded cDNAcDNAs, prior to the final library amplification.
To perform the qPCR assay you will need these reagents in addition to your library prep kit:
The PCR Add-on and Reamplification Kit V2 for Illumina (Cat. No. 020.96208)*, and
SYBR Green I nucleic acid stain (10,000X in DMSO, not provided in the kit).
*For QuantSeq-Pool, if you have >7 pools, the PCR Add-on and Reamplification Kit V2 is required for qPCR. If you have <6 pools, the provided kit solutions for Library Amplification (PM, P5, P7, PE) are sufficient for qPCR and final amplification.
ATTENTION: ! The use of SYBR Green I-containing qPCR mastermixes from other vendors is not recommended.
Prepare SYBR Green I 2.5x Working Stock
SYBR Green I nucleic acid stain is provided at a 10,000x concentrated stock. We recommend using Sigma-Aldrich, Cat. No. S9430 or ThermoFisher, Cat. No. S7585.
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The working stock can be aliquoted into smaller volumes and stored at -20 deg. Celciusdegrees Celsius.
qPCR Assay Protocol
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.First, 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. 
and Reamplification Kit V2 User Guide.