Both mutants have stable mutations in target genes and will be referred to as 13124R and NCTRR in this study. Both mutants had a mutation in gyrA (G81C, D87Y), 13124R had mutation in gyrB (A431S) and parC (S89I), and NCTRR this website had a mutation in parE (E486K).
The bacteria were grown anaerobically under an atmosphere of 85% N2, 10% CO2, and 5% H2 at 37°C in brain heart infusion (BHI) broth (Remel, Lenexa, KS) with vitamin K (1 μg/ml), hemin (5 μg/ml), and L-cysteine (5 μg/ml) (Sigma Chemical Co., St. Louis, MO) [29]. No antibiotics were added. Preparation of RNA Early exponential (2.5-3.0 h) growth phase cultures of all four strains, grown in BHI under identical anaerobic conditions, were used to isolate RNA for microarrays. Cells from 100-ml cultures were harvested by centrifugation (15,000 × g, 10 min, 4°C), washed with 10 mM Tris and 1 mM EDTA (pH 8.0), and suspended in 1 ml of buffer containing 10 mg/ml of lysozyme (Sigma).
The mixtures were incubated for 10 min at room temperature and centrifuged (15,000 × g, 10 min, 4°C). The samples were suspended in 0.5 ml TE (10 mM Tris, 1 mM EDTA) and mixed with 5 ml of RNA-Bee isolation reagent from TEL-TEST, Inc. (Friendship, TX). After addition of 1 ml chloroform to the mixture, the samples were incubated on ice for 30 min and centrifuged (15,000 × g, 30 min, 4°C). The clear phases were harvested, added RAD001 ic50 to an equal volume of isopropanol and centrifuged to pellet the RNA. The RNA was further purified using an RNeasyR Mini Kit (50) from QIAGEN, Inc.
(Valencia, CA), according to the instructions provided with the kit. After RNA extraction and purification, contaminating DNA was removed using 10 U of RNase-free DNase 1 (Boehringer Mannheim, Ingelheim, Sunitinib purchase Germany). The quantity and quality of total RNA was determined using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technology, Wilmington, DE). RNA purification steps for real-time PCR (qRT- PCR) were essentially the same. The RNA was stored at −80°C and used within a week to avoid degradation of RNA. RNA was extracted from three different cultures of each strain for microarray analysis and qRT-PCR. Probe design for microarrays The probes were designed by Biodiscovery LLC, Ann Arbor, MI (http://www.mycroarray.com/) from the sequences of C. perfringens strains 13 (CPE) and ATCC 13124 (CPF in http://www.ncbi.nlm.nih.gov), using OligoArray v 3.1 (http://berry.engin.umich.edu). The designs of microarrays were submitted to MIAMExpress and can be accessed at the following links: for strain 13124, [http://www.ebi.ac.uk/arrayexpress/arrays/A-MEXP-2008], and for strain NCTR, at [http://www.ebi.ac.uk/arrayexpress/arrays/A-MEXP-2027]. Microarray hybridization The microarrays were hybridized by Biodiscovery LLC to fluor-labeled RNA at 60°C for at least 16 h in 2-gasket slides and commercial hybridization chambers (Agilent, Santa Clara, CA) while being rotated (~4 rpm) in a hybridization incubator.