How many PCR cycles are needed to amplify QuantSeq libraries?

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.).

_{*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 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

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 and Reamplification Kit V2 (Cat. No. 208), 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 and Reamplification Kit V2 User Guide, as well as in the Appendices of each QuantSeq 3' mRNA-Seq Library Prep Kit User Guide.

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