Why do I need to do a qPCR assay for CORALL V2 libraries? How does the qPCR assay work?
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 while overcycling can lead to higher duplication levels and quantification biases.
The qPCR is particularly important for low RNA input amounts, FFPE and low or variable quality RNA input samples. 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 RNA 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 the CORALL RNA-Seq V2 Library Prep Kit:
PCR Add-on and Reamplification Kit V2 for Illumina (Cat. No. 208) – 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).
IMPORTANT: The use of SYBR Green-containing qPCR mastermixes or alternative fluorophores (e.g., EvaGreen) are explicitly NOT recommended as their performance can vary and influence the calculation of the number of PCR cycles required for the endpoint PCR. The SYBR concentrations may be inhibitory, and the buffer/enzymes included may alter the qPCR efficiency. Amplification curves generated using other fluorophores 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.
For more information on the qPCR assay, see this FAQ.
For more information on how to calculate the endpoint PCR cycle number from the qPCR assay, see this FAQ.
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.