Micro dismembrator s

Cell pellets were disrupted under frozen conditions with 3000 rpm for 1 min, using a Micro-Dismembrator S (Sartorius, Göttingen, Germany). Total RNA was isolated directly from freeze-milled preparations, using 1 ml QIAzol Lysis Reagent (Qiagen, Hilden, Germany). After addition of 0.2 ml chloroform (Sigma-Aldrich, Steinheim, Germany), samples were shaken and incubated at RT for 10 min. For phase separation, samples were centrifuged at 15000 g for 20 min at 4 °C and the aqueous phase was transferred to a fresh tube.
Total RNA precipitation was performed by mixing 0.5 ml Isopropanol (Sigma-Aldrich, Steinheim, Germany) with the aqueous phase. After incubation at RT for 10 min samples were centrifuged at 15000 g over night at 4 °C. RNA pellets were washed twice with 1 ml 75% Ethanol (Merck, Darmstadt, Germany) and centrifuged at 15000 g for 20 min at RT. After drying pellets, total RNA was dissolved in 32 μl RNAse free water (Gibco, Darmstadt, Germany). Concentration and purity was determined by Nanodrop (ND-1000, Thermo Fisher, Waltham, MA).

Total RNA was isolated using Micro-Dismembrator S (Sartorius, Germany) and RNeasy Mini Kit (Qiagen, Germany) in accordance with the manufacturer's instructions. For the detection of bacteria, DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany) and further treated with proteinase K in accordance with the manufacturer's protocol. Purified RNA and DNA were quantified using Qubit 2.0 fluorometer (Invitrogen, USA) and their quality was determined by Agilent Bioanalyzer 2100 (Agilent Technologies, USA). All RNA samples were treated with DNase I (Thermo Fisher Scientific, USA), and cDNA was synthesized using M-MLV Reverse Transcriptase (Thermo Fisher Scientific, USA) and random hexamers according to standard manufacturer's protocol.

For RNA extraction, deep-frozen kidney samples were homogenized in a Micro-Dismembrator S (Sartorius Stedim Biotech GmbH, Göttingen, Germany) at 3000 rpm for 30 s and resuspended in 1 mL of TRIZOL™ reagent (Sigma-Aldrich, Steinheim, Germany). RNA was extracted according to the manufacturer’s protocol and used for reverse transcriptase-polymerase chain reaction (RT-PCR) (RevertAid™ first-strand cDNA synthesis kit, Thermo Fisher Scientific, Darmstadt, Germany) using a random hexamer primer for amplification. Real-time PCR (TaqMan^®) was carried out with the Applied Biosystems 7500 Fast Real-Time PCR System. TaqMan^® gene expression assays and PCR Low Rox Mix were obtained from Thermo Fisher Scientific (Darmstadt, Germany). The TaqMan^® gene expression assays are listed in the Appendix A, Table A1 (Thermo Fisher Scientific, Darmstadt, Germany). The threshold cycle (Ct) was calculated by the instrument’s software (7500 Fast System SDS Software version 1.4). Analysis of the relative mRNA expression was performed using the ΔΔCt method. Eukaryotic 18S ribosomal RNA (Thermo Fisher Scientific, Darmstadt, Germany) served as a reference for normalization.