Quantitative PCR analysis of pre-amplified cDNA

The pre-amplified, ExoI-treated cDNA was diluted 1:5 prior to analysis through quantitative PCR (qPCR) with the microfluidic 96.96 Dynamic array Standard BioTools UK Ltd., London, UK) performed on a BioMark HD instrument (BioMark) (as described in [16 (link)]). Assay mixes were prepared by mixing 2.25 µL of 2X Assay Loading Reagent (Fluidigm), 2.5 µL of primer pair mix (1.15 µM) and 0.25 µL of low-EDTA TE buffer. Sample mixes were prepared by mixing 2.5 µL of TaqMan Gene Expression Master Mix (TFS), 0.25 µL of 20X EvaGreen DNA binding dye (Biotum), 0.25 µL of 20X GE Sample Loading reagent (Fluidigm) and 2 µL of pre-amplified, ExoI-treated cDNA (diluted 1:5). The thermal cycling conditions for qPCR were: thermal mix at 50 °C for 2 min, 70 °C for 30 min and 25 °C for 10 min, followed by a hot start step of 50 °C for 2 min, 95 °C for 10 min and then 30 cycles of 95 °C for 15 s and 60 °C for 60 s, with the fluorescence emission recorded after each cycling step. After the completion of the run, a melting curve of the amplified product was determined (60 °C for 3 s to 95 °C). Raw quantitation cycle (Cq) data were collated with the Real-Time PCR Analysis software v 3.1.3 (Fluidigm), setting the parameters of the quality threshold (0.65), baseline correction (derivative) and Cq threshold method to auto (global).