P. chrysosporium cDNA mixed with prairie soil cDNA, then biotin-labeled
Treatment protocol
After approximately 4 days of conditioning, ground wheat straw was added to the soils and the soils incubated another 7 days.
Growth protocol
Prior to RNA extraction, soils (50 g) were conditioned at room temperature and 0.03 MPa water content.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from soil using a mechanically-assisted (bead beating) method. Briefly, 0.5 g moist soil, 0.25 g 0.1-mm zirconia/silica beads, 0.3 mL 50 mM sodium acetate + 10 mM EDTA (pH 5.1), 0.3 mL 10% sodium dodecylsulphate, and 0.6 mL phenol:chloroform:isoamyl alcohol (25: 24:1) were added to a screw top tube. Cell lysis was accomplished by bead beating (Biospec products) for 2 min at homogenize speed, heating to 60ºC for 10 min, followed by an additional two min bead beating treatment. The RNA extract was centrifuged for 10 minutes at 15000 x g to collect the aqueous layer. RNA was purified from the aqueous phase by isopropanol precipitation, then resuspended in 50 µL nuclease free H2O and quantified on a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE.)
Label
biotin
Label protocol
Superscript II (Invitrogen Corporation, Carlsbad, CA) was used to generate first strand cDNA products from the soil RNA extract. Following cDNA generation, products were labeled with biotin (Bioprime, Invitrogen, Carlsbad, CA). In the first bioprime reaction, the entire cDNA reaction and 20µL of 2.5X Random Prime solution was mixed, denatured for 5 min at 95°C and snap cooled on ice. While the denatured cDNA mixture was on ice 1 µL of non-biotinylated 10 mM dNTP’s, 7 µL of water and 2 µL Klenow were mixed and incubated at 37°C for 4 hours. A second bioprime reaction was created by using a 10 µL aliquot of the first bioprime reaction as template. To the template 10 µL water, and 20 µL of 2.5X Random Prime solution were added, mixed, and denatured at 95°C for 5 min. Products were snap cooled and 5 µL 10x dNTP’s, 3 µL water, and 2 µL of Klenow were added, mixed, and incubated at 37°C for 4 hours. After incubation 5 µL stop buffer was added to stop polymerase function and the reaction was purified using a min-elute PCR purification kit (Qiagen, Inc., Valencia, CA).
Hybridization protocol
Bioprimed cDNA was hybridized to gene specific probes printed on epoxysilane coated 6-well microarray slides. The signal was amplified using an optimized TSA kit (Perkin Elmer, Waltham, MA) protocol. First, slides were pre-hybridized with TNB blocking buffer for 30 min at 22°C. The target cDNA was mixed with hybridization buffer (2X SSC, 0.02% Tween 20, and 1 ul per well of 1 ng µl-1 salmon sperm DNA) and denatured at 95°C for 2 min. After removal of TNB blocking buffer, the denatured cDNA was added to the microarray slide. Lip01 and mnp02 control DNA was added at a concentration of 100 ng/well, P. chrysosporium was added at 250 ng/well, and soil DNA was added at a concentration of 1000 ng/well in a 75 µl per well volume. Hybridizations were incubated for 3 hours in a humidified chamber at 21°C (plus or minus 1 degree), and then washed with TNT buffer for 2 min with agitation. The biotin in the biotin labeled target cDNA was amplified by incubating microarray slides with 1:100 SA-HRP (Strepavidin-horse radish peroxidase) in TNB for 30 min at 22°C. The SA-HRP was removed by washing slides in TNT buffer. Background biotin binding was reduced by incubating slides with 10% equine serum in 2X SSC for 30 min at 22°C. Slides were washed once more with TNT buffer and incubated with 1:50 Biotinyl Tyramide (amplification reagent in amplification diluent) for 10 min at 22°C and washed in TNT buffer. A SA-Alexa 546 (Molecular Probes, Invitrogen Corporation, Carlsbad, CA) fluorescent indicator (1:500 SA-Alexa in 1x SSC, 5X Denhardt’s solution) was added to each array to bind to the amplified biotin labeled target cDNA and incubated in the dark for 1 hour, at 22°C. Subsequent to fluorescent binding, slides were washed in TNT buffer, quickly dipped in 0.6X SSC, and dried with compressed nitrogen.
Scan protocol
ScanArray, Perkin Elmer, Waltham, MA
Description
P. chrysosporium cDNA mixed with prairie soil cDNA, then biotin-labeled
Data processing
The microarray spot intensities were extracted with an automated microarray image analysis toolbox for MatLAB (White, A. M., D. S. Daly, A. R. Willse, M. Protic, and D. P. Chandler. 2005. Automated Microarray Image Analysis Toolbox for MATLAB. Bioinformatics 21:3578-9.) Spot intensities were background corrected and log transformed prior to normalization and statistical analysis.
