DNA was extracted from 250 mg of rumen content following Yu and Morrison procedure (Yu and Morrison 2004). DNA yield and quality were determined after Nanodrop 1000 spectrophotometric quantification. DNA extracts were stored at -20°C until analysis.
Label
Cy3
Label protocol
DNA(2 microgrammes) was labeled using the ULS aRNA Labeling Kit and Cy3 cyanine (Kreatech Diagnostics, Amsterdam, Netherlands) following the manufacturer’s recommendations.
Hybridization protocol
DNA hybridization (1 microgram for each DNA) was performed at 65°C during 24 h following the Agilent protocol “Oligonucleotide Array-Based CGH for gDNA Analysis”.
Scan protocol
microarray slides were scanned using an Agilent G2505B microarray scanner (Agilent Technologies, Santa Clara, CA, United States) with a resolution of 5 microns and the XDR mode (Extended Dynamic Range) set up at 10 and 100% PhotoMultiplier Tubes (PMT) gain.
Description
rumen microbiota of young lambs
Data processing
Fluorescence intensities of oligonucleotide spots were extracted from the scanned images using the Feature Extraction software (V11.0, Agilent Technologies, Santa Clara, CA, United States). Data were processed with dedicated scripts based on C++ and Delphi languages. For each probe, median intensity value of the three replicates was conserved and used as the probe signal value. The signal-to-noise ratio (SNR), similar to the detection threshold response and corresponding to the probe signal value divided by the local background intensity value, was calculated for each probe. Results for 25- and 54-mer probes were treated independently and for each probe type, a gene was considered as detected when 65% of probes were positive. The SNR value of a detected gene was then calculated by the mean of the SNR of all 25- or 54-mer probes targeting this gene.
