Rotor gene rg 3000 real time pcr machine

Total RNA was harvested using TRIZOL reagent (Invitrogen) followed by DNase I treatment (Roche, Rotkreuz, Switzerland); 1.5 μg of total RNA was reverse transcribed using SuperScript Reverse Transcriptase III (Invitrogen) with random hexamers (Invitrogen) following the manufacturer’s instructions. The qPCR TBP forward and reverse primers for amplification are listed below. For quantitative real-time PCR, 2 μL of cDNA was analysed using a Rotor-Gene RG-3000 real-time PCR machine (Corbett Research, Sydney, Australia) with QuantiTect SYBR green (Qiagen, Manchester, UK). The cycling parameters were 95 °C for 15 min, followed by 45 cycles of 94 °C for 20 s, 58 °C–62 °C for 20 s and 72 °C for 20 s. TBP (F) TTC GGA GAG TTC TGG GAT TG TBP. (R) CTC ATG ATT ACC GCA GCA AA.

Total RNA was extracted from the epididymal adipose tissue using an Easy‐Blue kit (Intron Biotechnology Inc) according to the protocol provided by the manufacturer. Then, total RNA was quantified with a NanoDrop‐2000 (Thermo Fisher Scientific). cDNA was synthesized (an equal amount of total RNA) with the Moloney murine leukemia virus transcriptase and Oligo (dT) 15 primers (Promega) using a Life Touch thermal cycler (Life Eco, Bioer Technology). The program was set for 1 hr of initiation at 42°C, followed by 10 min of incubation at 95°C and 10 min at 4°C. RT‐PCR was performed using the QuantiTect SYBR Green PCR kit (Qiagen), according to the manufacturer's instructions. cDNA was amplified for 40 cycles of denaturation (95°C for 30 s), annealing (57°C for 40 s), and extension (72°C for 40 s) using a RotorGene RG3000 real‐time PCR machine (Corbett Research). The purity of the PCR product was determined using melting curve analysis. The relative quantification of the expression of each gene was calculated using the comparative threshold cycle (Ct) method (Applied Biosystems). mRNA levels were normalized to β‐actin. Primer sequences are shown in Table 2.

Total RNA was extracted from the epididymal adipose tissue using an Easy-Blue kit (Intron Biotechnology Inc., Seoul, Korea) according to the protocol provided by the manufacturer. Then, total RNA was quantified with a NanoDrop-2000 (Thermo Fisher Scientific, Wilmington, DE, USA). cDNA was synthesized (0.03 μg of total RNA) with the Moloney murine leukemia virus transcriptase and Oligo (dT) 15 primers (Promega, Medison, WI, USA) using a Life Touch thermal cycler (Life Eco, Bioer Technology, Hangzhou, China). The program was set for 1 h of initiation at 42 °C, followed by 10 min of incubation at 95 °C and 10 min at 4 °C. RT-PCR was performed using the QuantiTect SYBR Green PCR kit (Qiagen), according to the manufacturer’s instructions. The cDNA (20 μL) was amplified for 40 cycles of denaturation (95 °C for 30 s), annealing (57 °C for 40 s), and extension (72 °C for 40 s) using a RotorGene RG3000 real-time PCR machine (Corbett Research, Sydney, Australia). The purity of the PCR products was determined using melting curve analysis. The relative quantification of the expression of each gene was calculated using the comparative threshold cycle (Ct) method (Applied Biosystems, Foster City, CA, USA). mRNA levels were normalized to β-actin. Primer sequences are shown in Table 2.