Itag universal sybr green supermix

RNA was isolated from indicated MH-S cells. A 50-ng DNA-free RNA was used for the first strand of cDNA synthesis using a SuperScript III first-strand synthesis system (Life Technologies). Quantitative reverse transcription-PCR was performed using the iTag Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and gene-specific primers (Supplementary Table 1, synthesized in Integrated DNA Technologies, Coralville, IA, USA), in a CFX Connect Real-time PCR Detection System (Bio-Rad). Relative transcript levels were first normalized C_T values to GAPDH, and then normalized to the indicated control (2^−ΔΔCT).^{25 (link)}

Breast cancer PDXs were generated from one luminal (HCI-011) and four TN (HCI-001, HCI-002, HCI-004, HCI- 010) breast cancers serially passaged in NOD/SCID mice as described previously50 (link)51 (link). Tumour fragments were placed into cleared inguinal fat pads of pre-pubescent NOD/SCID mice, grown to 20–25 mm and subsequently dissected and stored by freezing in 90% FBS and 10% dimethylsulfoxide until used or fixed in paraffin for IHC. Total RNA was extracted according to the manufacturers' instructions using RNeasy Plus Mini kit (Qiagen, Hilden, MD, USA) and iScript Reverse Transcription Supermix for RT-QPCR (BIO-RAD) was used for cDNA synthesis. RT-QPCR was performed using iTAG Universal SYBR Green Supermix (BIO-RAD).

Approximately 50 mg of liver or intestinal scrapings were transferred into prefilled Lysis Matrix D micro-centrifuge tubes (MP Biomedicals, UK) containing 500 µl of Ribozol (Amresco LLC, Ohio, USA). The tissue was homogenized for 60 seconds at 6.0 m/s on a Fastprep-24^TM homogenizer (MP Biomedicals, UK) and total RNA was isolated in accordance with the manufacturer’s instructions (Ribozol, Amresco LLC, Ohio, USA). Total RNA (1 µg) was reverse transcribed into cDNA using the High Capacity cDNA reverse transcription Kit (Life Technologies, Paisley, UK) in accordance with the manufacturer’s instructions and real-time quantitative polymerase chain reaction (RT-qPCR) was performed on 10 ng cDNA using the iTag Universal SYBR Green SuperMix (Bio-Rad, Hertfordshire, UK) and 50 nM of each gene-specific oligonucleotide primer (See Supplementary Table S2): Initial melting (95 °C for 5 minutes) followed by 40 cycles of melting (94 °C for 15 seconds), annealing (60 °C for 15 seconds) and extension (72 °C for 30 seconds) was performed using a CFX Connect^TM Real-Time Instrument (Bio-Rad). Transcript levels in the HFD + P group were determined using 2^{−(ΔCt1–ΔCt2)}, where ΔCt represents the difference between the Ct for each target gene and β-Actin mRNA transcript levels and are expressed as a ratio of expression relative to the HFD group.