Taqman gene expression assays primers

DNA extraction was performed using QIAGEN Plasmid Plus Maxi Kits (QIAGEN, Cat. 12963, Hilden, Germany), RNA extraction was conducted using RNeasy Mini Kits (QIAGEN, Cat. 74104, Hilden, Germany), reverse-transcription was performed using High-Capacity cDNA Reverse Transcription Kits (Thermo Fisher Scientific, Cat. 4368814, Waltham, MA, USA) and quantitative PCR (qPCR) was performed using TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific, Cat. 4352042, Waltham, MA, USA) with TaqMan Gene Expression Assays primers (Thermo Fisher Scientific, Waltham, MA, USA) for human ADAM15 (Hs00187052_m1), AXL (Hs01064444_m1), and RPLP0 (Hs99999902_m1).

Cells were lysed and RNA was extracted using RNeasy Mini kit (Qiagen, 74106) according to manufacturer’s protocol. cDNA was prepared using the High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems, 4387406). RT-PCR reactions were performed using the TaqMan Gene Expression Master Mix (Thermo Fisher, 4369016). The following TaqMan Gene Expression Assays primers were obtained from Thermo Scientific: KRAS (Hs00364284_g1, 4331182, Reference sequence NM_004985.4, amplicon length 111), NRAS (Hs00180035_m1 S, 4331182, Reference sequence NM_002524.4, amplicon length 86), HRAS (Hs00978051_g1, 4331182, Reference sequence NM_001318054.1, amplicon length 63) and β Actin (4333762 F). Reactions were performed on an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher). Relative expression was quantified using the ΔΔCt method and using the average cycle threshold.

10 ng of RNA extracted from liver tissue was used for reverse transcription and hybridization reaction was performed using One step RNA-to-Ct (Thermo Fisher Scientific, Waltham, MA) kit and TaqMan gene expression Assays primers (Thermo Fisher Scientific, Waltham, MA) on ViiA7 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA) according to manufacturer’s instructions. The list of primers used for the quantitative RT-PCR is listed in Table 1. All values were normalized to GAPDH/Gapdh. All HSC samples were run in three biological replicates with three technical replicates per experiment. All mouse samples were run in 5 biological replicates with two technical replicates per experiment. Fold changes were calculated using 2^-ΔΔCt method. The calculated threshold values were determined by maximum curvature and ΔCt was calculated as Ct^control-Ct^sample.