Viia7 cycler

Total RNA was collected 4 days post lentiviral shRNA transduction using GENEzol TriRNA Pure Kit (Geneaid #GZX200). RNA concentration was measured using NanoDrop 1000 Spectrophotometer, and 1 µg of RNA was reverse transcribed to cDNA using SensiFAST cDNA Synthesis Kit (Bioline #65054). qPCR reactions were set up using PowerUp SYBR Green Master Mix (Applied Biosystems #A25742) and real-time detection and quantification of cDNAs was performed on the Viia7 Cycler (Applied Biosystems) with 40 cycles of amplification. Viia7 System Software (Applied Biosystems) was used to determine Ct values with automatically set thresholds. Gene expression was normalized to GAPDH and analyzed using the ΔΔCt method. The following RT-qPCR primers were used (h, human): hGAPDH, 5′-CTC CTG CAC CAC CAA CTG CT-3′ (forward), 5′-GGG CCA TCC ACA GTC TTC TG-3′ (reverse); hRAC1, 5′-CGGTGAATCTGGGCTTATGGGA-3′ (forward), 5′-GGAGGTTATATCCTTACCGTACG-3′ (reverse); hRAC2, 5′-CAGCCAATGTGATGGTGGACAG-3′ (forward), 5′-GGAGAAGCAGATGAGGAAGACG-3′ (reverse); hRAC3, 5′-ACAAGGACACCATTGAGCGGCT-3′ (forward), 5′-CCTCGTCAAACACTGTCTTCAGG-3′ (reverse); hCDC42SE2, 5′-GGATCAGGAGACCTGTTCAGTG-3′ (forward), 5′-CCTTCGTATCCACGAGCTGCAT-3′ (reverse); hPTCH1, 5′-GCTGCACTACTTCAGAGACTGG-3′ (forward), 5′-CACCAGGAGTTTGTAGGCAAGG-3′ (reverse).

qRT-PCR was performed essentially as described [69 (link)], except that RNA was isolated and purified using the High Pure RNA Isolation Kit (Roche Diagnostics) and RT-qPCR was carried out using 2X SYBR® Green PCR Master Mix (Bio-Rad Laboratories, Inc., Hercules, CA, USA) in a ViiA7 cycler (Applied Biosystems, Inc., Foster City, CA, USA). Primers for PCR were designed with IDT PrimerQuest software (Integrated DNA Technologies, Inc., Coralville, IA, USA): DCYTB forward 5′-TGCATACAGTACATTCCCGCCAGA-3′, DCYTB reverse 5′-ATGGAACCTCTTGCTCCCTGTTCA-3′, ACTB forward 5′-TTGCCGACAGGATGCAGAAGGA-3′, ACTB reverse 5′-AGGTGGACAGCGAGGCCAGGAT-3′. GREB1 primers were as described in [70 (link)].