Arsenic-treated plates were supplemented with 100 µM potassium arsenate (Sigma) according to a previously determined sub-lethal growth response curve.
Growth protocol
Arabidopsis thaliana ecotype Columbia seeds were surface sterilized and plated on agar-solidified MS culture medium supplemented with B5 vitamins, 10% sucrose, 2% Gelrite ®, pH 5.8. Plants were grown aseptically in petri plates that were cold stratified at 4°C for 24 hrs and then placed in a growth chamber at 25°C under a 16 hr photoperiod.
Extracted molecule
total RNA
Extraction protocol
After 10 d, 2 g of whole plant material (shoots + roots) was harvested from each plate, frozen in liquid nitrogen, and subjected to RNA isolation using Trizol ® reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s protocol.
Label
Cy3, Cy5
Label protocol
Total RNA from six biological replicates were purified using RNeasy MiniElute columns (Qiagen, Valencia, CA). A total of 1.25 µg of purified total RNA was subjected to Aminoallyl Message Amp II kit (Ambion, Austin, TX) first strand cDNA synthesis, second strand synthesis, and in vitro transcription for amplified RNA (aRNA) synthesis. aRNA was purified according to manufacturers protocol (Ambion, Austin, TX) and quantified using a Nanodrop spectrophotometer. Two 4 µg samples of aRNA were labeled with Cy3 and Cy5 monoreactive dyes (Amersham Pharmacia, Pittsburgh, PA) in order to conduct a dye swap technical replicate for each biological replicate. Each aRNA sample was brought to dryness in a Speedvac and dissolved in 5 µL of 0.2 M NaHCO3 buffer. Five microliters of Cy3 or Cy5 (in DMSO) was added to each sample and incubated for 2 hrs in the dark at RT. Labeled aRNA was purified according to kit instructions (Ambion, Austin, TX) and quantified using the Nanodrop spectrophotometer. One-hundred pmol Cy3- and Cy5-labeled aRNA targets were denatured by incubating at 65°C for 5 min and added to a hybridization mix containing 9 µl 20X SSC, 5.4 µl Liquid Block (Amersham Pharmacia, Pittsburgh, PA), and 3.6 µl 2% SDS for a 90 µl total volume.
Arsenic-treated plates were supplemented with 100 µM potassium arsenate (Sigma) according to a previously determined sub-lethal growth response curve.
Growth protocol
Arabidopsis thaliana ecotype Columbia seeds were surface sterilized and plated on agar-solidified MS culture medium supplemented with B5 vitamins, 10% sucrose, 2% Gelrite ®, pH 5.8. Plants were grown aseptically in petri plates that were cold stratified at 4°C for 24 hrs and then placed in a growth chamber at 25°C under a 16 hr photoperiod.
Extracted molecule
total RNA
Extraction protocol
After 10 d, 2 g of whole plant material (shoots + roots) was harvested from each plate, frozen in liquid nitrogen, and subjected to RNA isolation using Trizol ® reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s protocol.
Label
Cy5, Cy3
Label protocol
Total RNA from six biological replicates were purified using RNeasy MiniElute columns (Qiagen, Valencia, CA). A total of 1.25 µg of purified total RNA was subjected to Aminoallyl Message Amp II kit (Ambion, Austin, TX) first strand cDNA synthesis, second strand synthesis, and in vitro transcription for amplified RNA (aRNA) synthesis. aRNA was purified according to manufacturers protocol (Ambion, Austin, TX) and quantified using a Nanodrop spectrophotometer. Two 4 µg samples of aRNA were labeled with Cy3 and Cy5 monoreactive dyes (Amersham Pharmacia, Pittsburgh, PA) in order to conduct a dye swap technical replicate for each biological replicate. Each aRNA sample was brought to dryness in a Speedvac and dissolved in 5 µL of 0.2 M NaHCO3 buffer. Five microliters of Cy3 or Cy5 (in DMSO) was added to each sample and incubated for 2 hrs in the dark at RT. Labeled aRNA was purified according to kit instructions (Ambion, Austin, TX) and quantified using the Nanodrop spectrophotometer. One-hundred pmol Cy3- and Cy5-labeled aRNA targets were denatured by incubating at 65°C for 5 min and added to a hybridization mix containing 9 µl 20X SSC, 5.4 µl Liquid Block (Amersham Pharmacia, Pittsburgh, PA), and 3.6 µl 2% SDS for a 90 µl total volume.
Hybridization protocol
Microarrays comprised of 70-mer oligonucleotides obtained from the University of Arizona (http://ag.arizona.edu/microarray/) were immobilized by rehydrating the slide over a 50ºC waterbath for 10 s and snap drying on a 65ºC heating block for 5 s for a total of four times. Slides were UV-crosslinked at 180 mJ in a UV cross-linker (Stratagene, La Jolla, CA). The slides were then washed in 1% SDS, dipped in 100% EtOH five times followed by 3 min shaking. Slides were spun dry at 1000 rpm for 2 minutes and immediately placed in a light-proof box. The 90 µl hybridization mix was pipetted onto a microarray slide underneath a lifterslip (Lifterslip, Portsmouth, NH) and placed in a hybridization chamber (Corning, Corning, NY) overnight at 55ºC.
Scan protocol
After hybridization, slides were washed in 2X SSC, 0.5% SDS for 5 minutes at 55ºC, 0.5X SSC for 5 minutes at room temperature, and 0.05X SSC for 5 minutes at room temperature. Slides were then spun dry at 1000 rpm in a Sorvall centrifuge and scanned with a GenePix 4000B scanner (Axon Instruments, Inc., Union City, CA).
Description
condensed table of 6 hybridzations (slides 249, 247, 244, 250, 246, and 248)
Data processing
The intensity variation was removed by fitting a loess regression using SAS 9.1 (SAS, Cary, NC). Data were log-2 transformed and statistically analyzed using rank product statistics to identify differentially expressed genes. Bioconductor (www.bioconductor.org) Rank Prod package was used to perform the rank product analysis. Significantly different genes reported in this study exhibited P <0.001, as designated by the rank product analysis. The false discovery rate value obtained was based on 10,000 random permutations. Since 10,000 random permutations was very computer intensive, 1000 random permutations were performed 10 different times each time starting with a different random seed number and the average FDR value calculated was used for further analysis. The genes that had FDR values less than or equal to 0.01 were considered as differentially expressed.
