RNA extraction, cDNA synthesis and Taqman qPCR

Cell pellets are snap-frozen using dry ice.

RNA is extracted using the Maxwell® RSC simply RNACells kit (Promega), and the Maxwell® RSC 48 instrument, following manufacturer instructions.

After RNA extraction, RNA concentration and quality are measured and assessed using a nanodrop.

Up to 1 µg of RNA per sample is reverse-transcribed into cDNA using the High-Capacity cDNA Reverse Transcription kit (ThermoFisherScientific).

qPCR is performed using TaqMan^TMGene Expression Assay (Thermo Fisher Scientific) according to the manufacturer’s instructions.

For each gene, TaqMan^TMprobes were used along with the TaqMan^TMmaster mix, and sample cDNA following the manufacturer's protocol.

Samples, along with a minus reverse transcriptase control (-RT) were ran for each gene on the QuantSudio 6 Flex Real-Time PCR System (Applied Biosystems).

The -RT served as a negative control, and the gene expression levels were normalised to the housekeeping gene GAPDH following the delta-delta Ct method. Gene expression values were expressed as the normalisation to their respective control sample.