Quant studio 7 pcr machine

RNA was extracted from CRC cell lines and xenograft tumor tissues using the miRNeasy Mini Kit (Qiagen, Germantown, MD) following the manufacturer’s instructions. In brief, 3 mm cube xenograft tumor tissues were homogenized using TissueLyser II (Qiagen) with 5 mm stainless steel beads. RNA was isolated using Qiacube (Qiagen) and eluted in 50 µl of RNase-free water. Organoids were harvested in RLT lysis buffer and RNA was isolated with the RNeasy Mini Kit (Qiagen) using Qiacube and eluted in 50 µl of RNase-free water. Extracted RNA was used as a template for cDNA production using High Capacity cDNA Revers Transcription Kit (Thermo Fisher Scientific) according to manufacturer’s protocol. RT-qPCR was performed using SensiFAST SYBR mix (Bioline, London, UK) on Quant-Studio 7 PCR machine (Applied Biosystems). For specific primer sequences refer to Supplementary Table 1. All RT-qPCR target genes were calculated using ΔΔCt method normalized to β-actin. For miRNA expression analysis, we used the TaqMan real-time PCR assay kit (Applied Biosystems, Foster City, CA) and TaqMan microRNA Reverse Transcription Kit (Applied Biosystems) as described previously^{48 (link)}. For all reactions, TaqMan Universal Master Mix (Applied Biosystems) was used and the analysis was carried out using Quant-Studio 7 (Applied Biosystems). All data were analyzed using ΔΔCt method and normalized to RNU6B.

Thermal shift assays were carried out as described in (Niesen et al., 2007 (link)). Briefly, in a 96-well RT-PCR plate (Life Technologies) 1 μg HER3 kinase domain/well was incubated with 1 μM inhibitor or 200 μM ATP/10 mM MgCl₂ (as indicated) for 30 mins at 4°C in the presence of Sypro Orange dye (Sigma). HER2 TSA experiments were performed in a 384-well RT-PCR plate (Thermo Fisher Scientific). 0.5 μg of HER2 kinase domain/well was incubated with 1 μM lapatinib, 1 μM bosutinib, or 200 μM ATP/10 mM MgCl₂ for 20 mins at 4°C. HER3 measurements were taken on an Applied Biosystems 7500 Fast Real-Time PCR machine, and HER2 measurements on an Applied Biosystems Quant Studio 7 PCR machine. Data were trimmed and a Boltzmann sigmoidal curve fitted in GraphPad Prism 6. The inflection point of the Boltzmann sigmoidal was taken as the T_m. Thermal shift ΔT_m values were obtained by subtracting the T_m value of the kinase domain alone control.