7300ht fast real time pcr system

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), reverse transcribed using a reverse transcription kit (TaKaRa, PrimeScript™ RT reagent Kit), and normalized with GAPDH. A MiRNA Kit (Omega Bio-tex, R6842-01) was used to extract miRNA. Maxima SYBRGreen/ROX qPCR Master Mix (ZF102-2; ZOMANBIO, Beijing, China) was used for reverse transcription-quantitative PCR (RT-qPCR) to determine the relative quantification of miRNAs. MiRNAs were normalized with U6 snRNA (7300HT Fast Real-Time PCR system; Applied Biosystems, USA). The following primers were used: Pln forward 5′-CCAGTGAGCTTTCCTGCGTA-3′, reverse 5′-AGTTTGCAGGTCTGGAGTGG-3′; and Ank2 forward 5′-CAACTTTCTCGCCATGTCTGC-3′, reverse 5′-CCAAGAGTGACTGGGGTTTGA-3′. Each sample was analyzed in triplicates, and the analysis was performed using the 2^-ΔΔCt method.

The total RNA from the livers of WT and PASK-deficient mice under non-fasted, fasted and re-fed conditions was extracted with TRIzol (Life Technologies, Barcelona, Spain). RNA integrity was tested with the Bioanalyzer 2100 (Agilent), and cDNA synthesis was performed using the high-capacity cDNA archive kit (Applied Biosystems), using 2 µg of RNA as template, following the manufacturer’s instructions. Four microliters of a 1:10 dilution of the cDNA was used as a template for the polymerase chain reaction (PCR). Either TaqMan^® Assay (Applied Biosystems, Foster City, CA, USA) or SYBR Green^® Assay (Applied Biosystems) was used to quantify mRNA levels by RT-PCR in a 7300HT Fast Real-Time PCR System (Applied Biosystems). The details of the primers and probes are listed in Supplemental Table S1. The PCR conditions were 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. 18s and β-actin housekeeping genes were used for normalization. In the case of SYBR Green Assay, a standard curve was previously generated in each real-time PCR assay by tenfold serial dilutions of the cDNA samples.