7 protocols
Preprocessing and normalization followed a standard procedure using methods described by Smyth & Speed (Smyth & Speed, 2003)
Purified samples were then dried, before the dried samples were resuspended in 140 uL hybridization solution (5 SSC, 0.1 % (wt/vol) SDS, 1.0 % (wt/vol) bovine serum albumin, 50 % (vol/vol) formamide and 0.01 % (wt/vol) single-stranded salmon sperm DNA) and hybridized for 16h at 42 C to the E. faecalis oligonucleotide array in a Tecan HS 400 pro hybridization station (Tecan). Arrays were washed 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 x SSC and with filtrated H20. Two replicate hybridizations were conducted for each test strain. The Cy3 and Cy5 dyes (PerkinElmer) were swapped in one of the two replicate hybridizations. hyb temp (42 degree_C); hyb volume (135 uL)
cDNA was synthesized and labeled with the Fairplay III Microarray labeling kit (Stratagene) according to the manufacturer’s protocol, with the following modifications: For each labeling reaction, 10 µg of total RNA and 500 ng of random primers were initially preheated at 70°C for 10 min. A reverse transcription-PCR mixture (10x AffinityScript RT buffer, a 20x deoxynucleoside triphosphate mixture, 0.1 M dithiothreitol, 20 U RNase block, and AffinityScript HC RT) was added to the annealed primers and RNA, and the reaction mixture was further incubated for 3 h at 42 C. After labeling, 1 µL of hydroxylamine (Sigma Aldrich) was added to quench the coupling reaction, and the reaction mixture was incubated 10 min. at room temperature. 70 µL RNase-free water was then added, and unincorporated dyes were removed from the samples by using the QIAquick PCR purification kit (QIAGEN).
Total RNA was isolated by means of FastPrep (BIO101/Savent) as follows: The frozen cell pellets were thawed and 350µL lysis buffer RLT (RNeasy Mini Kit; Qiagen), supplemented with 0.1% (vol/vol) beta-mercaptoethanol (Sigma), was added to each cell suspension. The suspensions were transferred to tubes containing 0.6g acid-washed, RNase-free glass beads (< 106microns) (Sigma) and 250µL chloroform (Merck). Cells were lysed by shaking the tubes for 20s at speed 6 in a FastPrep bead-beater (BIO101/Savent). The tubes were centrifuged for 2min at 13000 rpm (Biofuge Pico, Heraeus), and supernatants transferred to eppendorf tubes. Pellets were resuspended in 350µL beta-mercaptoethanol-containing lysis buffer RLT, and the cell suspensions transferred back to the tubes with the glass beads for a second extraction, to increase the overall RNA yield. 250µL ethanol was added to the supernatant fluids from the two extractions, and after mixing, each sample was applied to an RNeasy spin column (Qiagen). The columns were further washed and RNA eluted according to the RNeasy Mini kit protocol (Qiagen), with on column-DNA digestions with RNase-free DNase I (Qiagen) according to the manufacturer’s recommendations.
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.
ON-cultures were diluted 1000x and grown at 37 degree_C in a defined medium to OD600 ~0.2, centrifuged and pellets flash frozen in liquid nitrogen.