As1480 maxwell rsc simply rna tissue kits

RNA extraction from tumor tissue and reverse transcription using previously described TaqMan PCR primers and probes were performed as previously described (21 (link)). Briefly, RNA was extracted from tumor tissue using the AS1480 Maxwell RSC Simply RNA tissue kits (Promega, USA) according to the manufacturer’s instructions. One microgram of RNA was reverse transcribed in cDNA using the High-capacity RNA cDNA kit (ThermoFisher Scientific, France) and amplified using the TaqMan Universal PCR Master Mix, No AmpErase UNG (ThermoFisher Scientific, France). Primers and probes specific for Gli1 target genes and control gene β-actin (Table S1) were purchased commercially and used according to the manufacturer’s instructions (ThermoFisher Scientific, catalog number 4331182). All values were normalized to the control gene β-actin using the ΔΔ Ct method. Here, again, a BCC sample was used as positive control.

RNA was extracted from tumor tissue using the AS1480 Maxwell RSC Simply RNA tissue kits (Promega, Madison, WI, USA) according to the manufacturer’s instructions. RNA concentrations were calculated using Multiscan GO reader, V.1.01.10 (ThermoFisher Scientific, Saclay, France). One μg RNA was reverse-transcribed in cDNA using the high-capacity RNA-cDNA kit (ThermoFisher Scientific, Saclay, France) according to the manufacturer’s instructions. cDNA amplification was performed using the TaqMan Universal PCR Master Mix, No AmpErase UNG (ThermoFisher Scientific, Saclay, France) according to the manufacturer’s instructions. TaqMan PCR primers and probes specific for Gli1 target genes and controls β-actin were purchased commercially and used as per the manufacturer’s instructions (ThermoFisher Scientific, catalog number 4331182) (Table S4). All values were normalized to the control gene β-actin using the ΔΔ Ct method. Two samples not expressing Gli1 as assessed by IHC were taken as reference samples or calibrators.