1xtaqman universal pcr master mix

cDNA was synthesized using a high capacity cDNA reverse transcription kit from Applied Biosystems. Quantitative real time PCR reactions were performed with 1x Taqman universal PCR master mix (Applied Biosystems) in a MicroAmp optical 96-well reaction plate. All reactions were done in triplicate using two dilutions (1/500, 1/1000) each in 20μl reaction mix consisting of 9μl of cDNA, 1μM of each primer, and 10μl Taqman mastermix. The plates were then covered tightly with an optical adhesive cover and centrifuged at 1400 x g for 30 seconds at 4°C. PCR reactions were run on an ABI Prism 7500 (Applied Biosystems) and results were analyzed using the same software. Probes and primers were purchased from Life Technologies. Relative gene expression levels were normalized to GAPDH/TBP.

Quantitative PCR was performed using the LightCycler 480 II (Roche), according to manufacturer’s instructions, using a mixture containing 45 ng cDNA, 1xTaqMan Universal PCR Master Mix, no AmpErase UNG (Applied Biosystems), 1xTaqMan SNP Genotyping Assay (Applied Biosystems), and nuclease-free water (Ambion) in a 20 μl reaction volume. ACTB (Applied Biosystems, cat#Hs99999903_m1) was included as reference gene. A standard curve was generated using pooled equal amounts of cDNA from all samples. Quantification of the dual-color hydrolysis of both allele-specific fluorescent reporter dyes, FAM (“G” allele) and VIC (“A” allele), was performed with the LightCycler 480 SW 1.5.1 software (Roche) using the 2^nd derivative method, according to manufacturer’s instructions.

Cells from the human hepatocellular carcinoma cell line HepG2 were plated in six-well culture dishes (Corning) containing 10% fetal bovine serum (FBS)-RPMI-1640 medium supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL). For each gene, cells were transfected with siRNAs (Ambion, Life Technologies, Additional file 2: Table S9), using Lipofectamine RnaiMax according to the manufacturer’s instructions (Invitrogen). Two days after transfection, siRNA-targeted cells and mock-treated controls (scramble siRNA) were incubated with oleic acid (0.75 mM, Sigma Aldrich,) for 24 h in 1% FBS medium. Thereafter, the cells were examined for effects on levels of PCSK9 and LDLR, and on measurements of cholesterol (total cholesterol, TC; and cholesteryl esters, CE) and triglycerides (TG).
Total RNA was isolated from HepG2 cells with the RNeasy Mini-kit (Qiagen) and concentrations determined by NanoDrop (Thermo Scientific). The efficiency of target gene silencing was determined by TaqMan analyses (Additional file 2: Table S9). In brief, cDNA was synthesized from 0.5 μg of total RNA (High-Capacity RNA-to-cDNA Kit, Thermo Scientific) and amplified by real-time PCR with 1xTaqMan universal PCR master mix (Applied Biosystems) using primers and probes from Applied Biosystems (Additional file 2: Table S9); data were normalized to mock control using the comparative Ct method.