Soil DNA was extracted following the instructions of the PowerMax Soil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA). DNA quality was assessed via NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA), based on UV spectrometry absorbance ratios at wavelengths of 260 nm/280 nm and 260 nm/230 nm. DNA concentration was measured by PicoGreen using the FLUOstar OPTIMA fluorescence plate reader (BMG Labtech, Jena, Germany) before microarray labeling.
Label
Cy3
Label protocol
One microgram of DNA from each sample was mixed with random primers and denatured before dNTP, fluorescent dye Cy-3 dUTP and DNA polymerase were added for labelling at 37 C for 6 h, followed by heating at 95 C for 3 min.
Hybridization protocol
For hybridization, DNA was resuspended in hybridization solution containing a sample tracking control, formamide, SSC, SDS, a Cy3-labelled alignment oligonucleotide, a Cy5-labelled alignment oligonucleotide and a Cy5-labelled common oligonucleotide reference standard target. After denaturing, the mixtures were deposited onto the glass microarray and hybridized at 42 °C for 16 h.
Scan protocol
Then the arrays were washed and dried, and scanned by an MS 200Microarray Scanner (NimbleGen) at 532nm and 635 nm.
Description
C5
Data processing
NimbleScan software version 2.5 (NimbleGen) was used to grid and process the images to transform them into signal intensity. The raw signals from NimbleScan were submitted to the Microarray Data Manager on our website (http://ieg.ou.edu/microarray), cleaned, normalized and analysed using the data analysis pipeline.
