Quantstudio 3 real time thermocycler

The transcripts of selected growth-related genes were quantified using quantitative PCR (qPCR) analysis with liver tissues. The RNA isolation was performed using TRIzol followed by DNA removal using TURBO DNA-free™ Kit and RNA cleaning with Qiagen RNeasy Mini Kit. The cDNA of each RNA sample was synthesized using the Invitrogen SuperScript IV Reverse Transcriptase system (Thermo-Fisher). The transcript levels of the genes encoding glutathione S-transferase (gst), glutathione peroxidase (gpx), glutathione-disulfide reductase (gsr), growth hormone receptor II (ghr2), catalase (cat), superoxide dismutase (sod), fatty acid synthase (fas), acetyl-CoA carboxylase β (acacb), and carnitine palmitoyltransferase 1 (cpt1) were analyzed using qPCR on a QuantStudio 3 Real-Time Thermocycler (Applied Biosystems, Waltham, MA) with the following cycling conditions: 50 °C for 2 min; 94 °C for 2 min; and 40 temperature cycles including 30 s of 94 °C, 30 s of 55 °C, and 30 s of 72 °C. The primers for qPCR are listed in Supplementary Data 1. The Ct values of all selected genes in all samples were normalized using the Ct values from a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (gapdh), and relative transcript levels of each gene in livers from the fish with different diets were calculated using the 2^-ΔΔCt method [32 (link)].

MCF-7 and AsPC-1 cells were seeded in 6-well plates at a density of 2.5 × 10⁵ cells/well for 24 h. The cells were treated with different concentrations of nsLTP1 (5, 10, and 20 μM) for 48 h. TRIzol reagent (Invitrogen, USA) was used to extract mRNA according to the manufacturer’s protocol. The RNA concentration was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). The yields were approximately 2 μg from each cell line which were reverse transcribed to cDNA using the RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific). The cDNA was used to quantify Bcl2, BAX, caspase-3, surviving, EGFR, and VEGF gene expressions by Real-Time Polymerase Chain Reaction (RT-PCR). Reactions were performed using Maxima SYBR Green/ROX qPCR master mix kit (Thermo Fisher Scientific) in QuantStudio 3 Real-Time thermocycler (Applied Biosystems, Thermo Fisher Scientific) under the following conditions: 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 55 °C for 60 s. GAPDH was used as the housekeeping gene. Fold change in gene expression was calculated using the 2^-ΔΔCt method and presented as the mean ± SD relative to control cells in three independent triplicate experiments [38 (link)].