Male P. deltoides 'Bart ex. Marsh', clone ILL-129 from central Illinois, USA
Biomaterial provider
Prof. Gail Taylor, University of Southampton
Treatment protocol
The fifth leaf below the first fully unfurled leaf was sampled from three replicate plants after 9 hrs exposure to ambient air in growth chambers. RNA was extracted from each leaf and then equal quantities of RNA from each replicate pooled to form a single sample for cDNA synthesis and hybridisation.
Growth protocol
Cuttings were planted in John Innes No. 2 compost (John Innes Manufacturers Association, Harrogate, UK) in 1.75 L pots with two buds above soil level. Pots were assigned randomly to two controlled environment rooms set at 22 ?C, PAR 160 µmol m-2s-1 and 16 hr photoperiod, and were watered daily. Thirty three days after planting, the plants were transferred to the ozone enrichment chambers. The growth chambers were described in detail by Warwick and Taylor (1995). Plants were acclimated for 48 hrs under continuous light before the ozone treatment was initiated, at a mean temperature of 24 °C and PAR 120 µmol m-2 s-1. Mean airflow through the chambers was 2.5 m s-1.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the fifth leaf of each plant using a modified version of the method described in Chang et al (1993): Spermidine was omitted from the extraction buffer, and 2.67 % ß-mercaptoethanol was added. After precipitation, two additional CHISAM extractions were performed. RNA concentration was determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany), and quality was assessed using a 1 % (w/v) agarose gel.
Label
Cy3
Label protocol
1 µl oligo dT (Invitrogen, Paisley, UK) was added to 25 µg total RNA in 15.5 µl RNase free water. The sample was heated at 65 °C in a heating block for 10 min and then was placed on ice for 1 min. A reverse transcription mastermix was prepared, consisting of 6 µl 5X first strand buffer, 3 µl DTT, 3 µl 10X amino-allyl dNTP mix, 0.5 µl Super RNase inhibitor and 1 µl SuperScript III per reaction (all from Invitrogen, Paisley, UK). 13.5 µl mastermix was then added to each sample, and the preparation incubated at 46 °C for 1 hr. 1 µl MMLV-RT RNase H point mutant (Promega, Southampton, UK) was added to each sample, and further incubated for 1 hr. 2 µl fresh 5M NaOH and 8 µl 0.5M EDTA were added and the samples incubated for 15 min at 65 °C to stop the reverse transcription and degrade remaining RNA. The reaction was neutralised with 20 µl 1M HEPES (pH 7.5). 40 µl water and 500 µl PB were added to each sample and mixed thoroughly. The sample was transferred to a QiaQuick column, and centrifuged at maximum speed for 1 min. The flow-through was discarded and 750 µl fresh phosphate–ethanol wash buffer added to the column. The column was centrifuged for 1 min and the flow through discarded. The column was then centrifuged for 1 min and transferred to a labelled 1.5 ml microfuge tube. 30 µl H-2O was added to the column, incubated at room temperature for 1 min and centrifuged for 1 min. A further 20 µl H¬2O was added and the column centrifuged for 1 min. The sample was dried in a speed vac (Savant, Minnesota, USA) to 27 µl. A tube of dye ester was re-suspended in 73 µl water-free DMSO, and 4.5 µl aliquots made, which were stored at -80°C. 3 µl 1M NaHCO3 (pH 9.3) were added to the cDNA sample. From this point onwards all steps are carried out in minimal light. 4.5 µl Cy-dye (Amersham, Buckinghamshire, UK) were added to the sample and incubated at room temperature for 1 hr. 4.5 µl 4M hydroxylamine were added to stop the reaction. The Cy3 and Cy5 reactions were mixed and 22 µl H2O added. The reaction was then dried in a speed-vac.
Male P. deltoides 'Bart ex. Marsh', clone ILL-129 from central Illinois, USA
Biomaterial provider
Prof. Gail Taylor, University of Southampton
Treatment protocol
The fifth leaf below the first fully unfurled leaf was sampled from three replicate plants after 9 hrs exposure to 200 ppb ozone in growth chambers. RNA was extracted from each leaf and then equal quantities of RNA from each replicate pooled to form a single sample for cDNA synthesis and hybridisation.
Growth protocol
Cuttings were planted in John Innes No. 2 compost (John Innes Manufacturers Association, Harrogate, UK) in 1.75 L pots with two buds above soil level. Pots were assigned randomly to two controlled environment rooms set at 22 ?C, PAR 160 µmol m-2s-1 and 16 hr photoperiod, and were watered daily. Thirty three days after planting, the plants were transferred to the ozone enrichment chambers. The growth chambers were described in detail by Warwick and Taylor (1995). Plants were acclimated for 48 hrs under continuous light before the ozone treatment was initiated, at a mean temperature of 24 °C and PAR 120 µmol m-2 s-1. Mean airflow through the chambers was 2.5 m s-1.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the fifth leaf of each plant using a modified version of the method described in Chang et al (1993): Spermidine was omitted from the extraction buffer, and 2.67 % ß-mercaptoethanol was added. After precipitation, two additional CHISAM extractions were performed. RNA concentration was determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany), and quality was assessed using a 1 % (w/v) agarose gel.
Label
Cy5
Label protocol
1 µl oligo dT (Invitrogen, Paisley, UK) was added to 25 µg total RNA in 15.5 µl RNase free water. The sample was heated at 65 °C in a heating block for 10 min and then was placed on ice for 1 min. A reverse transcription mastermix was prepared, consisting of 6 µl 5X first strand buffer, 3 µl DTT, 3 µl 10X amino-allyl dNTP mix, 0.5 µl Super RNase inhibitor and 1 µl SuperScript III per reaction (all from Invitrogen, Paisley, UK). 13.5 µl mastermix was then added to each sample, and the preparation incubated at 46 °C for 1 hr. 1 µl MMLV-RT RNase H point mutant (Promega, Southampton, UK) was added to each sample, and further incubated for 1 hr. 2 µl fresh 5M NaOH and 8 µl 0.5M EDTA were added and the samples incubated for 15 min at 65 °C to stop the reverse transcription and degrade remaining RNA. The reaction was neutralised with 20 µl 1M HEPES (pH 7.5). 40 µl water and 500 µl PB were added to each sample and mixed thoroughly. The sample was transferred to a QiaQuick column, and centrifuged at maximum speed for 1 min. The flow-through was discarded and 750 µl fresh phosphate–ethanol wash buffer added to the column. The column was centrifuged for 1 min and the flow through discarded. The column was then centrifuged for 1 min and transferred to a labelled 1.5 ml microfuge tube. 30 µl H-2O was added to the column, incubated at room temperature for 1 min and centrifuged for 1 min. A further 20 µl H¬2O was added and the column centrifuged for 1 min. The sample was dried in a speed vac (Savant, Minnesota, USA) to 27 µl. A tube of dye ester was re-suspended in 73 µl water-free DMSO, and 4.5 µl aliquots made, which were stored at -80°C. 3 µl 1M NaHCO3 (pH 9.3) were added to the cDNA sample. From this point onwards all steps are carried out in minimal light. 4.5 µl Cy-dye (Amersham, Buckinghamshire, UK) were added to the sample and incubated at room temperature for 1 hr. 4.5 µl 4M hydroxylamine were added to stop the reaction. The Cy3 and Cy5 reactions were mixed and 22 µl H2O added. The reaction was then dried in a speed-vac.
Hybridization protocol
1 L pre-hybridisation solution (5X SSC, 0.1% SDS, 2% BSA) was heated for 30 min at 50 °C in an oven. Slides was placed in a UV cross-linker (Stratagene, California, USA) set at 90 mJ and then washed for 30 s in 0.1% SDS, 30 s in MQ water and incubated for 3 min in near boiling water followed by 95% EtOH for 90 s, and centrifuged for 5 min at 500 rpm. Slides were immediately transferred to a staining dish containing pre-heated pre-hybridisation solution. The preparation was transferred to an oven and incubated at 50 °C. Slides were removed from the pre-hybridisation solution and washed for 30 s in MQ H2O and 30 s in 95% EtOH and centrifuged for 5 min at 500 rpm. A lifter slip (Erie Scientific, Portsmouth, USA) (pre-washed in 0.1% SDS, MQ H2O, and EtOH) was then placed on the spotted area of each slide. 2 µl poly-A, and 48 µl hybridisation buffer 3 (Ambion, Cambridgeshire, UK) were added to the probe and mixed. The probe was denatured by incubation at 96 °C for 2 min, and centrifuged at 13000 rpm for 30 s. The probe was then added to the slide adjacent to the lifter slip. Slides were incubated in a hybridisation chamber (Genetix, Hampshire, UK) at 50 °C for 16-18 hrs. Slides were then washed in coplain staining jars by gently agitated in wash buffer1 (2X SSC, 0.5% SDS) to remove the lifter slip and transferred to fresh wash buffer 1. The jar was incubated in an oven for 15 min at 50 °C. Slides were then transferred to wash buffer 2 (0.5X SSC, 0.5% SDS) for 15 min at 50 °C, wash buffer 3 (0.5X SSC) at room temperature for 5 min, WB4 (0.2X SSC) at room temperature for 1 min and WB5 (0.06X SSC) at room temperature for 1 min and then centrifuged for 5 min at 500 rpm.
Scan protocol
Slides were scanned using a ScanArray500 (PerkinElmer Life and Analytical Sciences, Boston, MA). Initially, a line scan was performed on a row of spots at 90 % laser power, and the Cy3 and Cy5 laser intensities equalised on the basis of this. The final scan was performed at 5 µm resolution, and the data saved in TIFF format.
Description
NA
Data processing
QC and normalisation was run in R using limma and sma function (R script linked as supplementary file).
