5 protocols
The labelled aRNA was then scanned on Agilent Technologies Scanner G2505B according to Two-Color Microarray-Based Gene Expression Analysis Protocol (Agilent).
Pairs of Cy3- and Cy-5 labelled aRNA samples were mixed together and hybridized to the microarray. Hybridization and post-hybridization washes were performed using Gene Expression Hybridization Kit (Agilent) and Gene Expression Wash Buffers (Agilent) according to the Bacterial RNA Amplification Protocol from Oxford Gene Technology company (Oxford, UK). In total, six independent hybridizations using RNA samples from three separate bacterial cultures were performed for the wild-type strain and both mutants.
Gene expresion profiles were performed by Oxford Gene Technology company (Oxford, UK) on microarray slides, consisting of 44,000 60-mer oligonucleotide probes covering the entire S. coelicolor genome. The whole steps from polyadenylation of total RNA to post-labelling of aminoallyl-containing aRNA were performed using MessageAmp II-Bacteria RNA Amplification Kit (Ambion) according to the Bacterial RNA Amplification Protocol from Oxford Gene Technology company (Oxford, UK). Briefly, purified total RNA was polyadenylated and subsequently reverse transcribed into first strand cDNA. After second strand cDNA synthesis, cDNA was purified and in vitro transcribed into amplified RNA (aRNA) in the presence of 5-(3-amionallyl)-UTP (Ambion). Modified aminoallyl-containing aRNA was purified and differentially post-labelled with Cy3 and Cy5 NHS-ester reactive dyes (GE Healthcare). Finally, labelled aRNA was purified using RNeasy MinElute clean up kit (Qiagen). Dye-swap labelling was performed for all labelled RNA samples.
Harvested mycelia were mixed with 0.5 ml of RNA Blue (Top-Bio) and disrupted with glass beads. Homogenate was mixed with 0.5% Sarcosyl (wt/vol) and incubated for 5 min at room temperature. Sample was shaken with 120 ml of chloroform and cetrifuged at 13,000g for 10 min at 4 °C. Water phase containing RNA was taken off and precipitated by 0.1 vol. of 3M CH3COONa pH 5.2/1 vol isopropanol on ice. RNA was recovered by centrifugation at 13,000g for 10 min at 4 °C and RNA sediment was washed with 1 ml of 70% ethanol (wt/vol). The crude total RNA was solubilized in water. Total RNA was treated with DNase I (Qiagen) and purified futher using RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol.
The wild type strain M145 and both mutant strains (delta wdpB and wdpC) were cultured on modified R3 solid medium at 30?C and mycelium was harvested at times, when the aberrant phenotype of mutants begun to manifest; i.e. at 48 hours of cultivation for the ?wdpB and at 64 hours for the ?wdpC mutant.