Biorad hard shell pcr plates

Total RNA was extracted from tumors or activated/differentiated CD8⁺ T cells using TRIzol (Thermo Fisher Scientific). After DNAse I treatment and RNA cleanup with the RNeasy kit (Qiagen), 1 μg of total RNA was reversed transcribed into cDNA using the Invitrogen SuperScript II reverse transcriptase kit according to manufacturer’s recommendations (Thermo Fisher Scientific). The BioRad hard-shell PCR Plates (BioRad) were used. Typically, 5-10 ng of cDNA was added to each well in triplicates along with 7.5μL of the primer mixture, which consisted of 0.5μL forward primer (10μM), 0.5μL reverse primer (10μM), 1.7μL PCR grade water, and 5μL of Roches LightCycler 480 SYBR Green I Master (Roche). The plate was centrifuged at 300 RCF for 1 minute and read in the BioRad CFX384 Real-Time System (BioRad). The qPCR settings: 95°C for 10:00 minutes, 95°C for 0:10 minutes, 62°C for 0:20 minutes, 72°C for 0:20 minutes (Read Plate), GOTO step 2 for 39 times, 92°C for 0:05 minutes, melt curve 65°C to 97°C, at increment of 0.5°C for 1:00 minute (Read Plate), 40°C for 0:10 minutes. Primers are listed in Table S3.

The Bio-Rad Hard-Shell PCR Plates (BioRad) were used. In each well, 2.5 μl of sample was added in triplicates along with 7.5 μl of the primer mixture, which consisted of 0.5 μl forward primer, 0.5 μl reverse primer, 1.7 μl PCR grade water, and 5 μl of Roche LightCycler 480 SYBR Green I Master (Roche). The plate was centrifuged at 300 RCF for 1 minute and read in the Bio-Rad CFX384 Real-Time System (BioRad). The qPCR settings were as follow: 95 °C for 10:00 minutes 95 °C for 0:10 minutes, 62 °C for 0:20 minutes, 72 °C for 0:20 minutes (Read Plate), GOTO step 2 for 39 times, 92 °C for 0:05 minutes, Melt Curve 65.0 °C to 97.0 °C at increment 0.5 °C for 1:00 minutes (Read Plate), 40 °C for 0:10 minutes.