Nanodrop nd 1000 system

Tissue samples (50–100 mg) were homogenized in 1 ml TriFast (Peqlab) in a Tissue Lyser (Qiagen) at 20 Hz for 2 × 3 min; 250 μl chloroform was added, and samples were vortexed and centrifuged for 15 min at 14,000 g. The supernatant was mixed with 70% ethanol (1/3 vol/vol) and RNA was extracted with a commercial RNA extraction kit (Nucleo Spin RNA kit; Macherey-Nagel) according to the manufacturer’s instructions. RNA purity and concentration were determined using the NanoDrop ND-1000 system (PeqLab). cDNA synthesis was performed with 400 ng of RNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) with random hexamer primers, following the manufacturer’s instructions. Relative mRNA levels were determined by quantitative RT-PCR using the TaqMan Gene Expression Assay Technique and the 7900HT Fast Real-Time PCR System (Applied Biosystems). The following assays were used: mTbp (Mm00446973_m1), mUcp1 (Mm00494069_m1), mAdrb3 (Mm00442669_m1), and mDio2 (Mm00515664_m1). Gene expression was normalized to the housekeeper TATA-box binding protein (mTbp) using the ΔΔC_T method.

For real-time quantitative RT-PCR (qRT-PCR), total RNA was isolated from the middle region of the cecum samples with 1,000 μl Trifast-GOLD reagent (PeqLab, Biotechnologie GmbH, Erlangen, Germany) according to the manufacturer's instructions. RNA quality and concentrations were determined using the NanoDrop ND-1000 system (PeqLab, Biotechnologie GmbH).
All details of the specific primers for the detection of expressed chB6 and IgA as well as the housekeeping gene 18S are described in Table 2. The specific primers for CD4 were obtained from Qiagen Quantitect primer assays. qRT-PCR was performed using the 7300 real-time PCR system (Applied Biosystems, Warrington, UK) with SYBR green as a double-stranded-DNA-specific fluorescent dye. The following cycle profile was applied: 1 cycle at 95°C for 2 min and 40 cycles at 95°C for 15 s, 59°C for 30 s, and 72°C for 30 s, followed by 1 cycle at 95°C for 15 s, 57°C for 30 s, and 95°C for 15 s.
The results were normalized to the housekeeping gene 18S (39 (link)); its expression was comparable between birds of C. jejuni-inoculated and noninoculated groups. Data are expressed as 40 delta threshold cycle (40-C_T) of mRNA expression in the tissues of C. jejuni-inoculated birds and C. jejuni-free controls.

Genomic DNA was prepared from 1070 whole blood samples. Extraction of DNA was performed using QIAamp DNA blood Mini kits or the Qiagen BioRobot EZ1 Workstation (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA concentration was determined using the Nanodrop ND-1000 system (PeqLab, Erlangen, Germany).