Kapa sybr pcr kit

cDNA synthesis was performed using 1–5 μg of total RNA with random hexamers and reverse transcriptase (GoScript, Promega, Charbonnières-les-Bains, France). Quantitative RT-QPCR was performed in triplicates for each samples using the KAPA SYBR PCR kit (KK4600, Sigma, Saint-Quentin-Fallavier, France) with a LightCycler 480 apparatus (Roche, Basel, Switzerland). 10–100 ng of cDNA was used for each reaction. Primers are listed in Table S2. Relative expression values were calculated using the 2^−ΔΔCT method and normalized using the β-actin gene. All QPCR reactions were performed with three independent cultures.

O4⁺-purified cells and LGG275 cells were infected with either a control-YFP or NICD-YFP virus (gift from Dr. Sutton, Yale, New Haven, CT, USA; multiplicity of infection = 6). NICD cDNA codes for amino acids from 1762 to 2556 of human NOTCH1 [24 (link)]. Five days after transduction, RNAs were extracted using the Arcturus Picopure RNA kit (Thermo Fisher), and 100–200 ng of cDNA was synthesized with random primers and reverse transcriptase (Promega, Charbonnières-les-Bains, France, GoScript). Quantitative PCR was performed using 2.5 ng of cDNA in duplicates. Primers (Sigma) are listed in Table S3. The KAPA SYBR PCR kit (Sigma) was used for QPCR (Light Cycler 480, Roche). Gene expression was calculated using the 2^−ΔΔCt method, using β-actin (ACTB) for normalization. For inhibitor experiments, LGG275 cells were treated with γ-secretase inhibitors (DAPT (GSI-IX, Selleck Chemicals, Breda, Netherlands) and LY411575 (Peprotech)) at 10 µM for 5 days before RNAs were extracted for QPCR. For the DLL4 ligand experiment (Figure S13A), plates were coated with DLL4 (5 µg/mL; Peprotech) or BSA at 4 °C overnight before seeding the cells. RNAs were extracted after 4 days. For BMP signalling experiments described in Figure 6C, cells were treated with 10 ng/mL of BMP2 or BMP4 (Peprotech) for 5 days with the addition of new cytokines every 2 days.

cDNA synthesis was performed using 1–5 μg of total RNA with random hexamers and reverse transcriptase (Promega, GoScript). Quantitative RT-qPCR was performed in triplicate for each tumour sample using the KAPA SYBR PCR kit (Sigma Aldrich KK4600) with a LightCycler 480 apparatus (Roche). 1 μL of cDNA was used for each reaction. Primers are listed in Table S2. Expression values were calculated using the 2^−ΔΔCT method and normalized using the housekeeping gene RPLP0.