Takara pcr thermal cycler mp

Semi-quantitative RT-PCR was performed as described previously [15 (link)]. Total RNA was isolated from cells using Trizol (BBI, Kitchener, Ont., Canada), and samples of total RNA were quantified by reading optical density at 260 nm. mRNA was reverse transcribed using a first strand cDNA synthesis kit (Fermentas, Glenn Burnie, Md., USA) according to the manufacturer’s instructions. One microgram of cDNA was amplified in the standard reaction mixture using 2× PCR MasterMix (TIANGEN, China) and the forward and reverse primers for hBD-2 and β-actin, an internal standard. The forward and reverse primers and product sizes were as follows: hBD-2 (255 bp) forward: 5′-CCAGCCATCAGCCATGAGGGT- 3′; reverse: 5′-GGAGCCCTTTCTGAATCCGCA- 3′. For β-actin (300 bp), forward: 5′-TCACCCACACTGTGCCCATCTACGA- 3′; reverse: 5′-CAGCGGAACCGCTCATTGCCAATGG- 3′. Amplification proceeded in a PCR Thermal Cycler (Takara PCR Thermal Cycler MP; Takara Bio, Otsu-shi, Japan) using 32 cycles consisting of 94 °C for 60 s, 62 °C for 30 s, and 72 °C for 120 s. The PCR products were verified by electrophoresis in a 1.2 % agarose gel and detected by ethidium bromide staining. Detectable fluorescent bands were visualized with an ultraviolet transill. The values of densitometry of the mRNA bands were acquired using ImageJ software (National Institutes of Health, USA) and then normalized with β-actin.

Differentiated THP-1 cells were treated with GGOH (10 μM) for 24 h and then treated with LPS (1 μg/mL) for 3 h. After the incubation, THP-1 cells were washed with phosphate-buffered saline (PBS) twice and then homogenized in guanidine isothiocyanate-based reagent, ISOGEN (Nippon Gene, Tokyo, Japan). Total RNA was isolated from the cells according to the manufacture’s manual. RNA integrity was analyzed by agarose gel electrophoresis, and RNA quantity was determined by its absorbance at 260 nm. Five micrograms of total RNA was used for cDNA synthesis. The RNA was denatured at 65 °C with oligo-dT/random primers and 10 mM dNTP. The mixture was then incubated in 50 mM Tris-HCl buffer (pH 8.3), 0.1 mM DTT, 50 units Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA), and 20 units RNaseOUT RNase inhibitor (Invitrogen) at 25 °C for 5 min, at 50 °C for 60 min, and finally at 70 °C for 15 min in a TaKaRa PCR Thermal cycler MP (Takara Bio). Aliquots of the synthesized cDNA were used as a template for quantitative PCR in the Applied Biosystems 7300 Real Time PCR System (Applied Biosystems, Foster City, CA, USA). The target cDNAs were amplified by gene-specific primers (Table 1) using the SYBR Premix Ex Taq solution (Takara Bio). The expression levels of the mRNAs were normalized to the level of eukaryotic translation elongation factor 1α1 (EEF1A1) mRNA [34 (link)].