phenotype: Not responsive to e[CO2] genotype: Clone 216
Treatment protocol
In this study, only plots receiving ambient air ([CO2] = 372 µL L-1) and air enriched with CO2 (target [CO2] = 560 µL L-1) were used. The site is divided into three blocks, each containing a CO2 and a control plot. Elevated CO2 plots were fumigated from sunrise to sunset for the length of the growing season (May-September), and one-minute averages of [CO2] in the e[CO2] plots were within 20% of the target concentration. Upper canopy sun lit leaves were harvested between 10:00 and 11:00 am on each date. Tissues were frozen in liquid nitrogen within 20 seconds of harvesting and stored at -80 oC until analyzed. To avoid potential variation derived from time-of-day effects, all pairs of control vs. e[CO2] samples were collected with 10 minutes of one another.
Growth protocol
The study was performed on leaf tissues collected near Rhinelander, WI, U.S.A. (89.5° W, 45.7° N) at the Aspen FACE experimental site, which has been operating since 1997. Twelve, 30m diameter, plots are contained within a 32 ha site in a full factorial design. Plots are exposed to ambient air (control) and air enriched with CO2, ozone, or CO2 and ozone in combination. Due to their significant differences in growth response to e[CO2], this study utilized two trembling aspen (P. tremuloides Michx.) genotypes, clone 216 and clone 271. Since bud-break in this region occurred on May 19 and bud-set occurred in late August (for clone 216) and early September (for clone 271), harvest dates of June 15th (early) and August 17th (late), 2005 were chosen to assure recently mature early-season leaf collections and mature but not yet senescing leaf collections in the late season time point.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted as described by Cseke et al. (2004), and treated with the TURBO DNA-free kit (Ambion Inc, Austin, TX) to remove genomic DNA contamination. • Handbook of Molecular and Cellular Methods in Biology and Medicine-2nd ed., L.J. Cseke, P.B. Kaufman, G.K. Podila, and C.J. Tsai: CRC Press, Boca Raton, Florida, 2004.
Label
Cy3
Label protocol
RNA (13.5 µg of each sample) was reverse transcribed using a SuperscriptTM Indirect cDNA Labeling System (Invitrogen, Carlsbad, CA), with oligo-dT primers. cDNA was purified using S.N.A.P. Columns (Invitrogen, Carlsbad, CA) and resuspended in 10 µl Coupling Buffer following the manufacturer’s instructions. Individual aliquots of Cy3 and Cy5 (RPN 5661, GE Healthcare, Piscataway, NJ) were resuspended in 5 µl DMSO, mixed with 5 µl of the cDNA in Coupling Buffer and incubated at room temperature for 1 h in the dark. The labeled cDNAs were purified using S.N.A.P. purification columns, and the yield and dye incorporation were evaluated using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). Targets were prepared by combining 40 pmol of each labeled sample in an amber microfuge tube. This was flash frozen in liquid nitrogen and dried in a vacuum centrifuge before storage at -20°C.
phenotype: Not responsive to e[CO2] genotype: Clone 216
Treatment protocol
In this study, only plots receiving ambient air ([CO2] = 372 µL L-1) and air enriched with CO2 (target [CO2] = 560 µL L-1) were used. The site is divided into three blocks, each containing a CO2 and a control plot. Elevated CO2 plots were fumigated from sunrise to sunset for the length of the growing season (May-September), and one-minute averages of [CO2] in the e[CO2] plots were within 20% of the target concentration. Upper canopy sun lit leaves were harvested between 10:00 and 11:00 am on each date. Tissues were frozen in liquid nitrogen within 20 seconds of harvesting and stored at -80 oC until analyzed. To avoid potential variation derived from time-of-day effects, all pairs of control vs. e[CO2] samples were collected with 10 minutes of one another.
Growth protocol
The study was performed on leaf tissues collected near Rhinelander, WI, U.S.A. (89.5° W, 45.7° N) at the Aspen FACE experimental site, which has been operating since 1997. Twelve, 30m diameter, plots are contained within a 32 ha site in a full factorial design. Plots are exposed to ambient air (control) and air enriched with CO2, ozone, or CO2 and ozone in combination. Due to their significant differences in growth response to e[CO2], this study utilized two trembling aspen (P. tremuloides Michx.) genotypes, clone 216 and clone 271. Since bud-break in this region occurred on May 19 and bud-set occurred in late August (for clone 216) and early September (for clone 271), harvest dates of June 15th (early) and August 17th (late), 2005 were chosen to assure recently mature early-season leaf collections and mature but not yet senescing leaf collections in the late season time point.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted as described by Cseke et al. (2004), and treated with the TURBO DNA-free kit (Ambion Inc, Austin, TX) to remove genomic DNA contamination. • Handbook of Molecular and Cellular Methods in Biology and Medicine-2nd ed., L.J. Cseke, P.B. Kaufman, G.K. Podila, and C.J. Tsai: CRC Press, Boca Raton, Florida, 2004.
Label
Cy5
Label protocol
RNA (13.5 µg of each sample) was reverse transcribed using a SuperscriptTM Indirect cDNA Labeling System (Invitrogen, Carlsbad, CA), with oligo-dT primers. cDNA was purified using S.N.A.P. Columns (Invitrogen, Carlsbad, CA) and resuspended in 10 µl Coupling Buffer following the manufacturer’s instructions. Individual aliquots of Cy3 and Cy5 (RPN 5661, GE Healthcare, Piscataway, NJ) were resuspended in 5 µl DMSO, mixed with 5 µl of the cDNA in Coupling Buffer and incubated at room temperature for 1 h in the dark. The labeled cDNAs were purified using S.N.A.P. purification columns, and the yield and dye incorporation were evaluated using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). Targets were prepared by combining 40 pmol of each labeled sample in an amber microfuge tube. This was flash frozen in liquid nitrogen and dried in a vacuum centrifuge before storage at -20°C.
Hybridization protocol
All microarray slides were checked using a dissecting microscope for uniformity of spots. Slides were prehybridized for 30 min on a slow rotary shaker in 5X SSC, 0.1% SDS, and 1% BSA, and rinsed 20 times in ddH2O. DNA was denatured by incubating slides in 95°C ddH2O for 1 min, followed by washing in 100% ice-cold ethanol for 15 s. Slides were dried by centrifugation (4°C, 2500 rpm, 2 min) and stored at 4°C. Hybridization was performed within 1 h of prehybridization. Targets were resuspended in 55 µl hybridization solution (50% formamide, 5X SSC, 0.1% SDS, and 0.1% BSA) and centrifuged at 14,000 × g for 1 min before denaturation at 45°C for 5-10 min. Hybridization was performed in Corning hybridization chambers (Corning Inc., Acton, MA) with Lifter slips (Erie Scientific Co., Portsmouth, NH) and incubated in a 43°C water bath in a hybridization oven for 36 h. Slides were rinsed in wash solution I (1X SSC, 0.2% SDS), prewarmed to 45°C, to remove the lifter slips. Slides were washed once in wash solution I for 15 min at 45°C with gentle agitation, once in wash solution II (0.1X SSC, 0.2% SDS) for 10 min, and twice in wash solution III (0.1X SSC) for 2 min each. Finally, slides were immersed in ddH2O for 10 seconds, 100% ethanol for 10 seconds, then dried by centrifugation (4°C, 2500 rpm, 2 min) and stored in darkness at room temperature.
Scan protocol
Slides were scanned using a VersArray ChipReader™ scanner (BioRad, Hercules, CA) at 5 µm resolution. Cy3 and Cy5 images were aligned and spots flagged using VersArray Analyzer 5.0 (BioRad, CA).
Description
Ring 2.1 vs. 2.2
Data processing
After local background subtraction and exclusion of flagged spots, signal intensity was log(2) transformed and normalized by the LOWESS algorithm with a smoothing parameter of 0.2, using GeneGazer software (Bio-Rad, CA). Spots having intensities below 20 in both channels were excluded.
