Soil DNA was extracted from a 0.5g soil sub-sample using the MoBio DNA Power Soil kit (MoBio Laboratories, Carlsbad, CA).
Label
Cy5
Label protocol
Hybridizations were carried out for all samples (N=38) with 100 ng of genomic DNA labeled with Cy5 using the BioPrime Array CGH Genomic Labeling kit (Invitrogen, Carlsbad, CA).
Soil DNA was extracted from a 0.5g soil sub-sample using the MoBio DNA Power Soil kit (MoBio Laboratories, Carlsbad, CA).
Label
Cy3
Label protocol
Hybridizations were carried out for all samples (N=38) with 100 ng of genomic DNA labeled with Cy5 using the BioPrime Array CGH Genomic Labeling kit (Invitrogen, Carlsbad, CA).
Hybridization protocol
The concentrated labeled DNA was resuspended in a final volume of 30 µL of DIG Easy Hyb hybridization buffer (Roche Diagnostics, Laval, Quebec) containing 10 µg each of salmon sperm DNA (Sigma-Aldrich, St-Louis, MO) and tRNA (Sigma-Aldrich). Lambda genomic DNA, used as PC, was labeled with Cy3 and 100 ng of this Cy3-lambda (PC) was added to each sample probe labeled with Cy5, before the hybridization. The hybridization mixture was heated at 95°C for 2 min to denature the DNA, and after a quick spin was introduced by capillarity under the LifterSlip covering the pre-hybridized microarray. The slides were incubated at 50°C for 16-20 h in a water bath within hybridization chambers. The slides were washed three times in 0.1X SSC and 0.1% SDS for 10 min at 50°C with agitation in a Belly Dancer (Stovall Life Science Inc., Greensboro, NC). Then the slides were rinsed three times in 0.1X SSC, three times in isopropanol, and dried by spinning in a slide microspin.
Scan protocol
The DNA microarray slides were scanned in a ScanArray Lite (Perkin Elmer, Boston, MA) upgraded with ScanArray Express (version 2.2.0.22) at 10 µm resolution. Lasers at 543 nm and 633 nm were used to excite the Cy3 and Cy5 dyes, respectively. The resulting 16-bit TIFF files were quantified using ScanArray Express.
Description
experimental sample compared to reference DNA (lambda phage)
Data processing
The intensity of each spot was measured with ScanArray Express using the histogram quantitation method; the percentile ranges for histograms were 80% for low signal range, 95% for high signal range, 5% for low background range and 20% for high background range. The median intensity minus the median background intensity was used for further calculations performed in Microsoft Excel. The background measurement refers to the local spot background intensity. Prior to normalization, all spots with a Signal-to-Noise ratio (SNR) lower than 3 were removed. The SNR was calculated using the following formula: (Median Signal – Median Background)/Standard Deviation of Background. Normalization between each array was performed using an internal labeling control (IC) consisting of a 290 bp luxA fragment. This fragment was added to each labeling reaction at a ratio of 0.05% of the DNA to be labeled. The intensity of each Cy5-sample gene spot was corrected using the average intensity of all Cy3-lambda signals. Corrected intensity values were averaged over triplicate spots to give a single value per gene. The intensity of the genes for which hybridization of the probes could not be visually detected in image analysis was set to zero. Relative intensity values were then calculated by dividing the intensity of a gene by the sum of the intensity for all genes (excluding controls). This “relative abundance” represents the fraction of the total intensity that is due to a particular gene and this value was subsequently used in statistical analyses.
