NOTE: If you previously determined the optional cycle number for Endpoint PCR with CORALL V1 for a specific sample type / input amount, you can use the same cycle number with CORALL V2.
The qPCR assay is recommended to determine the optimal number of PCR cycles for the library amplification. This will avoid under or overcycling. Undercycling may result in insufficient library for sequencing, and overcycling can lead to higher duplication levels and quantification biases.
The qPCR is particularly important for FFPE and low or variable quality RNA input samples, and low RNA input amounts. Please be aware that different sample types (species, tissues, cell types), or the use of different upstream RNA processing steps (i.e. enrichment and selection methods) may affect the mRNA or non rRNA content of the sample. Therefore, more or less PCR cycles may be required, even if input amounts between sample types are consistent.
To perform the qPCR assay, the following reagents should be purchased along with you the CORALL Total RNA-Seq Library Prep Kit:
PCR Add-on Kit for Illumina (Cat. No. 020.96) – Available from Lexogen here.
SYBR Green I Nucleic Acid Stain (10,000x in DMSO) – Available from Sigma- (Cat. No. S9430) or ThermoFisher (Cat. No. S7585).
NOTE: The use of SYBR Green-containing qPCR mastermixes is explicitly NOT recommended. The SYBR concentrations may be inhibitory, and the buffer/enzymes included may alter the qPCR efficiency. This may lead to inhibition of amplification in general, or inaccurate endpoint cycle number calculations.
The use of EvaGreen dye at final concentrations of 0.1x – 1x is not officially recommended, as it is not fully compatible with the specified qPCR assay for cycle number calculation. Amplification curves generated using EvaGreen may be delayed and show reduced maximal fluorescence intensity compared to SYBR Green I dye. Therefore, endpoint cycle numbers may need to be calculated differently and verified by Endpoint PCR testing.
Passive reference dyes such as ROX are not required and ideally should not be included in qPCR mastermixes.
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