Collection of Steele's lab, Food Science, UW-Madison
Extracted molecule
total RNA
Extraction protocol
Cultures were mixed by vortexing with two volumes of RNAprotect reagent (Qiagen Inc., Valencia, Calif.) containing 10 mg/ml of rifampicin (Sigma, St. Louis, Mo.) and incubated for 5 min at room temperature, followed by centrifugation at 5,500 x g, 15 min, 4oC. The cell pellets were resuspended in 5 ml of lysozyme solution (20 mg/ml) containing 0.1 mg/ml of rifampicin and incubated 25 min at 37oC. After repeated centrifugation, the pellets were resuspended by vortexing in 5 ml of TRIzol reagent (Invitrogen, Carlsbad, Calif.) and incubated 10 min at room temperature. Next, 1 ml of chloroform was added followed by vortexing for 15 sec and incubation for 10 min at room temperature. The mixtures were centrifuged at 16000 ґ g for 20 min at 4oC, then 2.5 ml of the upper aqueous phase were transferred into a fresh tube, mixed with 2.5 ml of isopropanol, incubated at room temperature for 10 min and RNA was pelleted at 16000 x g, 20 min, 4oC. After washing in 75% ethanol, the RNA pellet was dried and dissolved in 100 μl of water. The isolated total RNA was treated with 5 U of RQ1 DNAseI (Promega, Madison, Wis.), then purified using the RNeasy purification system (Qiagen).
Label
biotin-N6-ddATP; Cy-3 post-hybe
Label protocol
cDNA was synthesized from 12 μg of total RNA using random hexamer primers (Amersham Bioscience, Piscataway, N.J.) and SuperScript II reverse transcriptase (Invitrogen). After the synthesis, template RNA was digested with RNaseH (Promega) and RNaseA (Epicentre, Madison, Wisc.), and cDNA was purified using the QIAquick PCR purification kit (Qiagen), then fragmented to approx. 70-base fragments using appropriately diluted RQ1 DNaseI (Promega). Fragmentation efficiency was determined with Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, Calif.). Fragmented cDNA was end-labeled with biotin-N6-ddATP (NEN/Perkin-Elmer, Boston, Mass.) using terminal deoxynucleotidyl transferase (Promega) followed by concentration of labeled-sample on Microcon YM-10 columns (Millipore, Bedford, Mass.).
Hybridization protocol
All hybridizations, staining and processing of arrays were performed essentially as described by Ulijasz et al., 2003 (44) by personnel at NimbleGen Systems. In brief: hybridizations were performed at 45oC for 16 hours on a rotisserie-like apparatus (HybriWheel) to enhance uniformity of hybridizations across the array surface. After hybridization, the arrays were washed in buffers of varying stringency, then streptavidin was conjugated to the end-labeled biotin, followed by biotin-anti-streptavidin in the presence of normal goat IgG, and finally conjugated to a Cy3-streptavidin.
Scan protocol
Arrays were scanned using an Axon model 4000 scanner (Molecular Devices Corporation, Union City, Calif.) and the data extracted using NimbleScan software.
Description
References: 1. Ulijasz, A.T., D. R. Andes, J. D. Glasner, B. Weisblum. 2004. Regulation of iron transport in Streptococcus pneumoniae by RitR, an orphan response regulator. J. Bacteriol. 186:8123-8136. 2. Bolstad, B. M., R. A. Irizarry, M. Astrand, and T. P. Speed. 2003. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics. 19:185-193.
Data processing
Array normalization was performed using a quantile normalization method of Bolstad et al., 2003
