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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.).
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ATTENTION!: These cycle numbers

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_{*Input range for QuantSeq V2 3’ mRNA-Seq FWD is 1 - 500 ng of total RNA. Cycle numbers for QuantSeq Flex depend entirely on the targeting strategy and should always be optimized using the qPCR Assay.}

ATTENTION! The cycle number ranges above are not recommendations for the given input amounts for Endpoint PCR! These ranges are provided as a reference only and will not suit all sample types! guideline only for choosing an approximate number of cycles for the Endpoint PCR Test. The ranges account for the 3 extra cycles needed when using 1.7 µl of cDNA for the Endpoint PCR Test, versus 17 µl (10x more) that is used for standard Endpoint PCR.

qPCR Assay for Endpoint PCR Cycle Number Determination

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To perform the qPCR assay you will need:

• The PCR Add-on and Reamplification Kit for Illumina V2 (Cat. No. 020.96208), 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.

Protocol

The recommended protocol for the qPCR assay is provided in the PCR Add-on Kit Instruction Manualand Reamplification Kit V2 User Guide, 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

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Additional Resources

For additional information on the qPCR Assay consult the following online FAQs:

https://faqs.lexogen.com/faq/what-do-i-need-for-the-qpcr-assay

https://faqs.lexogen.com/faq/how-do-i-calculate-endpoint-cycle-numbers