Sensifast lo rox kit

A total of 2.0 μg DNA, including pHT1 and/or empty pEF.Bos vector (see above), was transfected using Lipofectamine 3000 (Invitrogen) in 8.0E+05 293T cells. After 48 hrs, the cells were washed using PBS, lysed using TRIzol (Invitrogen), and RNA was purified using DirectZol RNA kit (zymo research) followed by DNase I treatment (Turbo DNA free Ambion). 500 ng RNA was used for reverse transcription using Superscript III First Strand synthesis system (Invitrogen) with 1 μL random hexamers (Invitrogen) and 1 μL RNAseOUT (Invitrogen). Resulting cDNA was then used for HEXIM1 and GAPDH qPCR using sensiFAST Lo Rox kit (Bioline) and the following primers: 5’-CACCAGCGATGACGACTT-3’ (forward) and 5’-TCATGTTCTGCAGGCTCT-3’ (reverse) for HEXIM1, 5’-ACCACAGTCCATGCCATCAC-3’ (forward) and 5’-TCCACCACCCTGTTGCTGTA-3’ (reverse) for GAPDH. Each condition was tested in triplicate experiments, and results are shown as mean HEXIM1 mRNA count relative to GAPDH, normalized to the mean relative count in the control condition (pHT1 = 0 ng/mL).

Liver samples (n = 4) from each treatment were taken into clean Eppendorf tubes and directly to liquid nitrogen then stored at –80 °C. Total RNA was extracted using TRIsure™ Kit (Sensi-Fast LO-ROX kit, Bioline, #94002. UK). The quality of RNA was verified on 1.5% agarose gel electrophoresis. The RNA samples were reverse transcribed to cDNA using the SensiFAST™ cDNA synthesis kit (Bioline, United Kingdom).
As presented in Table 2, primer sequences were used to amplify fatty acid synthetase (FAS) and lipoprotein lipase (LPL) according to Jiang, et al. [26 (link)]. Real-time was performed in Stratagene MX300P Real-time PCR (Agilent Technologies, Santa Clara, USA) machine, using the SensiFast™ SYBR Lo-Rox kit (Bioline, United Kingdom) following the manufacturer’s guidelines. The PCR protocol was as following: initial denaturation at 95 °C for fifteen min, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. Melting curve analyses were run to ensure a single product of each reaction. The values of Ct of the target genes were first normalized against Ct of the house-keeping gene (β-actin). These were then used to calculate the relative gene expression of the target gene as a fold change based on the Livak Method [27 (link)], where fold change equal 2^−∆∆CT.