tissue: top 8 cm of the topmost fully expanded leaf of four control plants stress: control
Treatment protocol
8 cm of the top most fully expanded leaf was harvested directly into liquid nitrogen and stored at -80 until RNA extraction.
Growth protocol
Corn was planted on May 10, 2008 at South Dakota State Research Farm about 15 miles east of Brookings, SD. The soil parent materials were loess over glacial outwash, and the soil series was Brandt silty clay loam (fine-silty, mixed, superactive, frigid Calcic Hapludolls) (Clay et al. 2009). The water content at planting ranged from 22-24%, and soil pH ranged from 6.0-6.5. Soil N at the beginning of the season was 44.5 (2008) and 41 (2009) kg N ha-1, respectively, from residual nitrate and legume credit. The experimental design was a randomized complete block design with four replications. A commercially available 97-d corn hybrid that had glyphosate-resistance and corn rootworm/corn borer stacked traits was planted on May 10, 2008, and April 28, 2009. The seeding rate was about 79,000 seeds ha-1. Row spacing was 76 cm. Plant matrerial was harvested on July 2, 2008. Weather conditions at harvest were partly cloudy with temperatures in the low to mid 80sF, with an 18 mph wind.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Trizol reagent5 and purified using Qiagen RNeasy MinElute cleanup kit6, following the manufacturer’s protocol.
Label
Alexa 555
Label protocol
First strand cDNA synthesis was performed using 1900 ng total RNA and second strand cDNA synthesis was performed using the resulting first-strand cDNA sample to make double-stranded cDNA using the Aminoallyl Message Amp II kit7. aRNA was synthesized using the resulting double-stranded cDNA. Technical replicates from each treatment were labeled with Alexa Fluor 6478 or Alexa Fluor 5558 dye. An Alexa Fluor 647-labeled sample from one treatment was mixed with an Alexa Fluor 555-labeled sample from another treatment.
Hybridization protocol
46,000-element microarray chip developed by the University of Arizona was hybridized using their protocol (International Microarray Workshop Handbook, 2009; Gardiner et al. 2005).
Scan protocol
Intensities based on fluorescence for each probe were visualized and quantified with a GenePix scanner9 and GenePix Pro software.
Description
080609 312dR 304dB.txt Alexa 555: Field experiments were conducted in 2008 at South Dakota State Research Farm about 15 miles east of Brookings, SD. The soil parent materials were loess over glacial outwash, and the soil series was Brandt silty clay loam (fine-silty, mixed, superactive, frigid Calcic Hapludolls (Clay et al. 2009. The water content at planting ranged from 22-24%, and soil pH ranged from 6.0-6.5. Soil N at the beginning of the season was 44.5 kg N ha-1, from residual nitrate and legume credit. Nitrogen application of 236 kg N ha-1 was applied at the beginning of the season. Herbicides were applied at V2 to control unwanted species and all plots were maintained weed-free by hoeing or hand-pulling if plants survived or if new plants emerged after initial treatment. The experimental design was a randomized complete block design with three replications. A commercially available 97-d corn hybrid that had glyphosate-resistance and corn rootworm/corn borer stacked traits was planted on May 10, 2008. The seeding rate was about 79,000 seeds ha-1. Row spacing was 76 cm. Plants were grown to the V8 stage of growth and harvested on July 2, 2008. Weather conditions at harvest were partly cloudy with temperatures in the low to mid 80sF, with an 18 mph wind.
Data processing
Data from dual channel hybridization was treated as if it were from single channel hybridizations. Dual channel hybn: shade removed at V8 rep 3 and control rep 3
GeneMaths XT software was used to log transform (log 2) the intensity readings and samples from each individual hybridizations were normalize against each other using default parameters in Genemaths XT software (data was treated as if it were from single channel hybridizations). Probes that had hybridization intensity less than 2 times the standard deviation plus the average of the negative controls were deleted (Horvath et al., 2007) and technical replicates for each probe were averaged to reduce any dye bias that existed. GeneMaths XT software was then used to identify P values based on ANOVA and individual t tests between treatments. Probes were considered differentially expressed if P values for any test were ≤0.05.
