Tissueruptor homogenizer

Total bacterial DNA was isolated from the intestinal content. Approximately 100 mg of material was lysed and purified using a Genomic Mini AX Stool (A&A Biotechnology, Gdynia, Poland), according to manufacturer’s instruction. Next, isolated DNA was evaluated using the NanoDrop 2000 (Thermo Scientific Nanodrop Products, Wilmington, USA) and agarose gel electrophoresis. The evaluated DNA was diluted to a working concentration of 2 ng/μl and stored at 4°C prior to further analyses.
Total RNA was isolated from the mucosal scrapings sampled from the chicken intestines. About 100 mg of the mucosal scrapings were first homogenized in 1 ml of Trizol (Invitrogen, Carlsbad, USA) using a TissueRuptor homogenizer (Qiagen GmbH, Hilden, Germany). RNA was purified from the lysate using a Universal RNA Purification Kit (EURx, Gdańsk, Poland), according to manufacturer’s instruction. All RNA samples were evaluated using the NanoDrop 2000 (Thermo Scientific Nanodrop Products, Wilmington, USA) and agarose gel electrophoresis. Additionally, 10% of the RNA samples were evaluated on an Agilent 2100 Bioanalyzer using an Agilent RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA). RNA isolates were kept frozen at -20°C.

Total RNA was extracted from samples using RNeasy mini kits (Qiagen). Briefly, frozen samples of rat femoral muscle were homogenized in 300 μL of RLT Buffer by Tissue Ruptor homogenizer (Qiagen). Then, 590 μL of Nuclease-Free Water (Ambion) and 10 μL of Qiagen Proteinase K solution were added. Homogenates were incubated at 55°C for 10 min and centrifuged for 3 min at 14000 rpm. The following part of protocol was performed as described by the manufacturer. RNA was quantified using a Pico Drop spectrophotometer (Picodrop Limited, UK). The quality of RNA samples was analyzed by measuring the ratio of absorptions at 260/280 nm. The purified total RNA was immediately used for cDNA synthesis or stored at −80°C. Generation of cDNA was performed with QuantiTect Reverse Transcription Kit (Qiagen) according to the protocol of the manufacturer, with 1 μg of total RNA used as starting material. Reverse transcription was performed in conditions optimized for use with this kit (25°C for 10 min, 37°C for 120 min, and 85°C for 5 min). The cDNA samples were kept frozen at −20°C.

Total RNA was isolated from the spleen. Fragments of the spleen tissue were homogenized with the TissueRuptor homogenizer (Qiagen GmbH, Hilden, Germany) in TRIzol^® LS Reagent (Ambion/Thermo Fisher Scientific, Valtham, MA, USA). Further steps of RNA isolation and purification were performed with a commercial kit (Universal RNA Purification Kit, EURx, Gdansk, Poland). RNA quality and quantity were evaluated by using electrophoresis in 2% agarose gel and a NanoDrop 2000 spectrophotometer (Scientific Nanodrop Products, Wilmington, NC, USA).