8 protocols
Bacterial cells were grown in BHI (brain heart infusion media), aerobic growth; without shaking at 37 (degree_C).
Bacterial cell harvesting: ON-cultures were diluted 50x and incubated in BHI to OD600 ~0.2, centrifuged and pellets were washed in TE-buffer (10mM Tris-HCl, 1mM EDTA) and quick frozen in liquid nitrogen.
Bacterial cell lysate was obtained after lysozyme digestion (10 ug/ml). Total RNA was extracted using RNeasy Mini kit (Qiagen) and residual DNA was removed on-column with RNase-free DNase I (27 Kunitz units, Qiagen), according to the manufacturers protocol and recommendations.
Hybridization: Purified probes were dried and resuspended in 140 ul hybridization solution(5× SSC, 0.1 % (wt/vol) SDS, 1.0 % (wt/vol) bovine serum albumin, 50 % (vol/vol) formamideand 0.01 % (wt/vol) single-stranded salmon sperm DNA) and hybridized for 16 hours at 42 Cto the Enterococcus faecalis oligonucleotide array in a Tecan HS 400 pro hybridizationstation (Tecan). Washing of the arrays were performed twice at 42 C with 2 x SSC + 0.2 %SDS, and twice at 23 C with 2 x SSC, followed by more stringent washes at 23 C with 0.2 xSSC and with filtrated H20.
Bacterial treatment and cell harvesting: ON-cultures were diluted 1:100 and incubated in BHI to OD600 =0.15, then the cultures were split in two. For the following 30 min incubation, the control culture was added 0.02 ml 1 M NaCl (the elution buffer used for pediocin purification), while the test culture was added a concentration of pediocin PA-1 of 40 x MIC in a volume of 0.02 ml 1M NaCl. Cell pellets were harvested by centrifugation and immediately frozen in liquid nitrogen and stored at -80C until RNA isolation.
CDNa was synthesized and labeled using the Fairplay III Microarray labeling kit(Stratagene) according to the manufacturer's protocol, with the following modifications:10 ug of total RNA and 500 ng of random hexamer as primers were initially denaturated at 70 C for 10 min, and then on ice for 5 min. The annealed template and primers were added a reverse transcription-PCR mixture (10x AffinityScript RT buffer, a 20x deoxynucleoside triphosphate mixture, 0.1 M dithiothreitol, 20 RNase block, and AffinityScript HC RT, in total 20 ul), and synthesis were performed for 3 h at 42 C. Quenching of the coupling reactions were preformed after adding 1 ul of hydroxylamine (Sigma Aldrich) and incubated 10 min at room temperature. Unincorporated dyes were removed from the reactions by adding70 ul RNase-free water and by using the QIAquick PCR purification kit (QIAGEN), and the purified probes were isolated.
Normalization of arraydata: Preprocessing and normalization followed a standard procedure using methods described by Smyth & Speed (Smyth & Speed, 2003). The normalized files this represent the transformed results, for each treatment group
Scanning: Hybridized arrays were scanned at wavelengths of 532 nm (Cy3) and 635 nm (Cy5) with a Tecan scanner LS (Tecan). Fluorescent intensities and spot morphologies were analyzed using GenePix Pro 6.0 (Molecular Devices), and spots were excluded based on slide or morphology abnormalities.