Hs00168352 m1

Total RNA was isolated from human hepatocytes and snap‐frozen mouse liver with an RNeasy Mini Kit (Qiagen). cDNA was synthesized with a high‐capacity cDNA Reverse Transcription Kit (Applied Biosystems) and random primers. The mRNA expression levels of genes of interest were analyzed via TaqMan real‐time PCR in a ViiATM7 System (Applied Biosystems). The TaqMan Gene Expression assays used were Mm01263610_m1 (for mouse Pcsk9), Mm00662319_m1 (mouse Fasn), Mm00443090_m1 (mouse Pklr), Mm00504420_m1 (mouse Pnpla3), Mm01282499_m1 (mouse Hmgcr), Hs00545399_m1 (human PCSK9), Hs01005622_m1 (human FASN), Hs00176075_m1 (human PKLR), Hs00228747_m1 (human PNPLA3), and Hs00168352_m1 (human HMGCR) (all from Applied Biosystems). Hprt (mouse Mm03024075_m1) and GADPH (human Hs02758991_g1) (Applied Biosystems) were used as internal controls.

Following treatment for 24 hours and 48 hours with an equivalent dose of atorvastatin per cell line as used for microarray experiments, the statin-induced feedback response for four selected genes included in our cholesterol signature (HMGCR, HMGCS1, MVD, and INSIG1) was validated in four breast cancer cell lines using RT-qPCR. Briefly, total RNA was reverse-transcribed into cDNA (High-Capacity cDNA Reverse Transcription Kit, Thermo Fisher Scientific) which was used as a template in the qPCR reaction. Pre-designed primers and hydrolysis probe assays were used to amplify the genes of interest (Applied Biosystems: HMGCR; HS00168352_M1, HMGCS1; HS00266810_m1, MVD; HS00964563_g1, INSIG1; HS01650979_m1, and ACTB; HS99999903_m1). Expression measurements were then made by comparing cycle-threshold (CT) of the amplicons of interest to an internal reference amplicon in the house-keeping gene ACTB. All samples were run in triplicate and no template controls were included in each run. Data was analysed using the 2 (-DeltaDeltaC(T)) method [30 (link)]. The final gene expression measurements represent the mean and s.d. of three biological replicates, each composed of at least two independent experiments.