Nanodrop nd 1000a uv vis spectrophotometer

The bacterial DNA was extracted from colon and caecum digesta and purified using the QIAamp DNA Stool Mini Kit (No. 51504; Qiagen, West Sussex, UK) as described by Castillo et al. [37 (link)]. Briefly, each frozen digesta sample (0.3 ~ 0.5 g) was thawed and homogenized in the InhibitEX buffer (Qiagen, West Sussex, UK) and centrifuged to obtain the supernatant fluid. After the addition of proteinase K (Qiagen, West Sussex, UK) to the supernatant fluid, the solution was mixed by vortexing and then centrifuged to collect the supernatant fluid. The latter was mixed with ethanol (96–100%), and DNA was purified by using the QIAamp spin column (Qiagen, West Sussex, UK). Total DNA was quantified using the NanoDrop^® ND-1000A UV-VIS spectrophotometer (Thermo Scientific, Wilmington, DE, USA) at an OD of 260 nm, and its purity was assessed by determining the OD260nm/OD280nm ratio. All of the samples had an OD260nm/OD280nm ratio of 1.7 to 1.9. The length of the genomic DNA in each sample was determined using 1% denatured agarose gel electrophoresis. The microbial DNA was stored at −20 °C until qPCR analysis.

Total RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR) was conducted as described by Guo et al [17 (link)]. Briefly, total RNA of hepatic and cardiac samples was isolated using TRIzol Reagent protocol (Invitrogen, Carlsbad, CA, USA). The RNA was quantified by using the NanoDrop ND-1000A UV-VIS spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and its purity was assessed by using 1% denatured agarose gel electrophoresis. One microgram of total RNA was reverse transcribed using a PrimeScript RT reagent kit with gDNA Eraser (Takara, Dalian, China) according to the manufacturer’s instruction. The cDNA was synthesized and stored at −20°C. The qRT-PCR assay was performed using the SYBR Premix Ex Taq (Takara, China) on an Applied Biosystems 7500 Fast Real-Time PCR System (Foster City, CA, USA). The primer sequences are listed in Table 2. To ensure the sensitivity and accuracy, both hepatic and cardiac samples were normalized internally using the average cycle threshold (Ct) of glyceraldehyde-3-phosphate dehydrogenase, which acted as an internal reference in each sample to avoid any artifacts from variation in the targeted genes. Data were calculated using the 2^−ΔΔCt method. Each biological sample was run in triplicate.

Each frozen sample (approximately 100 mg) was powdered and homogenized, and total RNA was isolated using the Trizol Reagent protocol (Invitrogen, Carlsbad, CA). Total RNA was quantified using the NanoDrop ^® ND-1000A UV-VIS spectrophotometer (Thermo Scientific, Wilmington, DE, USA) at an OD of 260 nm, and its purity was assessed by determining the OD260/OD280 ratio (1.8 to 2.1). Total RNA was treated with gDNA Eraser (5 min at 42°C) to remove genomic DNA and removal of gDNA was confirmed by PCR using treated or untreated RNA samples as templates for a comparison. Reverse transcription was then carried out using a PrimeScript ^® RT reagent kit (Takara, Dalian, China) according to the manufacturer’s instruction. In brief, the total volume of the cDNA reaction was 20 μL. The reaction mixture contained 10.0 µL RNA, 1.0 µL PrimeScript RT Enzyme Mix 1, 4.0 µL RT Primer Mix, 4.0 µL 5 × PrimeScript Buffer 2, and 1.0 µL RNase Free dH₂O. The cDNA reaction was performed under the following conditions (two-step amplification): 37°C for 15 min and then 85°C for 5 sec. Finally, the synthesized cDNA was stored at -20°C until use.