Moloney murine leukemia virus reverse transcriptase

The oligonucleotides and the probe used for initial screening of IBV were described previously [31 (link)]. These oligonucleotides and the probe target the relatively conserved IBV N gene at nucleotide positions 741–1077 of the IBV Massachusetts H120 reference strain (GenBank accession number: AM260960). RNase free water and Newcastle disease virus strain (HB1) were included as negative controls. One-step RT-PCR was performed with 12.5 μl of 2× RT-PCR buffer mix, 0.5 μlof MgSO₄ (50 mmol/l), 0.5 μl of Rox reference dye (25 mmol/l, Life Technologies), 0.5 μl of Moloneymurine leukemia virus reverse transcriptase (200 U/μl), 0.5 μlof Taq DNA polymerase (Life Technologies), 0.5 μl of primers (0.2 μmol/l), 0.25 μl of probe (0.1 μmol/l), and 5 μl of RNA template to make a final volume of 25 μl.The reaction was carried out using a StepOne Plus real-time PCR system (Smart Cycler, Cepheid, Sunnyvale, CA) at 50 °C for 15 min, 95 °C for 5 min, and followed by 40 cycles of 9 °C for 15 s; 60 °C for 45 s; 72 °C for 30 s, and a final extension step of 74 °C for 5 min. Amplifications were recorded and analyzed, and the threshold cycle (C_t) was determined with StepOne software (Smart Cycler).

Total RNA was isolated from ~100 mg frozen tissue samples using 1.0 ml TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. The RNA was treated with DNase I (Life Technologies) at 37 °C for 25–30 min, then purified by phenol/chloroform extraction and stored at −80 ºC. RNA quantity and quality were analysed by spectrophotometer readings. DNA contamination was checked by PCR using primers specific for genomic DNA. RNA integrity numbers were determined using an Agilent 2100 bioanalyser (Agilent Technologies, Santa Clara, CA, USA).
First-strand complementary DNA was synthesised from 2 μg RNA using 100 units Moloney Murine Leukemia Virus reverse transcriptase with 2.5 μM random decamers and 2.5 μM oligo dT primers (Life Technologies), 0.5 mM of each dNTP and 20 units RNase inhibitor in 1 × reverse transcriptase buffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂ and 5 mM dithiothreitol) in a final volume of 20 μl. The reaction was incubated at 44 °C for 1 h then inactivated at 92 °C, and the product was stored at −20 °C.

TRI reagent (Sigma, St. Louis, MO, USA), NanoDrop (NanoDrop Technologies, Wilmington, DE, USA), and Moloney-Murine Leukemia Virus reverse transcriptase (Life Technologies, Bleiswijk, The Netherlands) were used to extract and reverse transcribe total RNA from the liver and distal small intestine, respectively. The RNeasy Lipid Tissue Mini Kit (QI-AGEN Sciences, Germantown, MD, USA) was used to extract total RNA from brown adipose tissue (BAT), which was subsequently quantified via NanoDrop and reverse transcribed as described previously. On a StepOnePlusTM Real-Time PCR system (Applied Biosystems, Foster City, CA, USA), real-time quantitative PCR analyses were performed. Cyclophilin and 36b4 (Rplp0) were used as housekeeping genes for liver and intestine (Cyclophilin) and BAT (36b4). Gene expression levels were first normalized to the housekeeping genes and then to the mean of the corresponding control group.

Total RNA was isolated from liver and small intestine using TRI reagent (Sigma, St. Louis, MO), quantified by NanoDrop (NanoDrop Technologies, Wilmington, DE) and reverse transcribed using Moloney-Murine Leukemia Virus reverse transcriptase (Life Technologies, Bleiswijk, the Netherlands). RNA from brown adipose tissue (BAT) and subcutaneous white adipose tissue (scWAT) was isolated using an RNeasy Lipid Tissue Mini Kit (Qiagen, Venlo, the Netherlands) quantified by NanoDrop and reverse transcribed as detailed apreviously. Real-time quantitative PCR analyses were performed on a Step One Plus™ Real-Time PCR system (Applied Biosystems, Foster City, CA). Gene expression levels were normalized to cyclophilin as a housekeeping gene for liver and intestine and 36B4 (Rplp0) as a housekeeping gene for BAT and scWAT. Data were then further normalized to the mean of the respective control group.