Sybr green detection format

The transcript levels of the seven genes (Usp30, Grb10, Pld1, St6galnac5, Oxct2b, Khk, and Acsl4) were determined in the liver of the three generations of rats from the restricted and control groups. Each generation/diet group was composed of six female fetuses from day 19 of pregnancy. The total RNA was isolated using Trizol (Roche) reagent, following the standard protocol. The quality and quantity of RNA were checked on a Nanodrop spectrophotometer. Reverse transcription was performed using High Fidelity cDNA Synthesis Kit (Roche) following real-time PCR on a Light Cycler 480 Instrument (Roche) using SYBR Green detection format (Roche). Each sample was analyzed in duplicate and the results were normalized using two reference genes—Hprt (hypoxanthine-guanine phosphoribosyltransferase) and Tbp (TATA box binding protein)—while relative transcript quantification was performed using the 2-ΔΔCT method, according to Livak and Schmittgen [12 (link)]. The details of the primer sequences and amplicon lengths are given in Table B in S2 File.

The relative transcript level was measured for the genes involved in DNA methylation and histone modification pathways, namely: Dnmt1 (DNA Methyltransferase 1), Dnmt3a (DNA Methyltransferase 3 Alpha), Dnmt3b (DNA Methyltransferase 3 Beta), Mecp2 (Methyl–CpG Binding Protein 2), Hdac1 (Histone Deacetylase 1), Sin3a (SIN Transcription Regulator Family Member A). The total RNA was extracted from the liver, femoral muscle, and visceral white adipose tissues using Trizol (Roche) reagent and following the standard protocol. The quality and quantity of the RNA isolates were checked on a Nanodrop system. cDNA synthesis was performed using 1 μg of RNA and iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR was conducted on a Light Cycler 480 Instrument (Roche) using the SYBR Green detection format (Roche). The analysis was performed in duplicate for each sample (six females in each generation/age/diet group). We used two genes as internal control: Hprt (hypoxanthine-guanine phosphoribosyltransferase gene) and Tbp (TATA box binding protein gene). All details concerning primers and amplicon length are shown in Table B in S1 File. Relative transcript quantification was performed using the 2^-ΔΔCT method, following Livak and Schmittgen [15 (link)].