Arabidopsis thaliana, ecotype Columbia (Col-0). Age = 4 weeks. Developmental stage = mid rosette growth. Tissue = rosette.
Biomaterial provider
M.R. Roberts, Lancaster University, UK
Treatment protocol
Plants were left to acclimatise for 30 min in controlled environment growth cabinets at 22º C in the dark, with relative humidity between 55-60%. Plants were then wounded in the dark under a 15 watt Kodak safe light with a yellow 0B filter (PAR - 0 µmol m-2 s-1). Rosette leaves were mechanically wounded using a metal haemostat, once or twice dependent on size, so that a wound site is present at any point on the leaf within a 1cm radius. Leaves were wounded closest to the largest width of the leaf and perpendicular to the mid-vein. Wounded plants were returned to the same controlled environment for a further 60 min in the dark, after which whole rosettes were cut off at the base of the stem and frozen in liquid nitrogen.
Growth protocol
Surface-sterilised seeds were grown on half-strength Murashige and Skoog basal salt mixture, adjusted to pH 5.8 with 1 M KOH, with 1% sucrose and 0.6% plant cell tissue culture grade agar, in a controlled environment growth room following a 10/14 hour light/dark regime with PAR of 250 µmol m-2 s-1 and temperatures of 20 ± 2º C. Ten-day-old seedlings were subsequently transplanted to a sieved, pre-autoclaved compost and sand mixture (3:1) and plants grown in individual pots (Aracon system) in the same controlled environment growth room. Plants were watered daily by spraying.
Extracted molecule
total RNA
Extraction protocol
For each treatment, a single RNA extract was made from 15-17 pooled individuals. Frozen plant tissue was ground to a fine powder in a pre-cooled pestle and mortar with liquid nitrogen and acid-washed sand, and transferred to a 15 mL polypropylene centrifuge tube. 2 ml well mixed RNA extraction buffer (1:1 mix of phenol and 0.1 M LiCl, 0.1 M Tris.Cl pH 8.0, 10 mM EDTA and 1% SDS) at 95ºC was added per gram of fresh weight plant material and tubes vortexed until thoroughly mixed. Half a volume of chloroform/isoamyl alcohol (24:1) was added and mixed well by vortexing. After centrifugation in a bench top centrifuge at 3500 x g for 10 min at 4ºC, the upper aqueous phase was removed to a new 15 ml centrifuge tube and re-extracted with chloroform/isoamyl alcohol before centrifugation for a further 10 min as before. The aqueous phase was removed to a new centrifuge tube and mixed with an equal volume of 4 M LiCl and incubated overnight at 4ºC. Tubes were then centrifuged for 15 min at 3500 x g at 4ºC to pellet precipitated RNA. The supernatant was removed and the pellet drained and resuspended in 300 µl 0.3 M NaOAc pH 5.2, before being transferred to an Eppendorf tube. RNA was re-precipitated by the addition of 750 µl cold ethanol, followed by centrifugation in a microcentrifuge for 10 min at 14000 rpm. Pellets were drained and rinsed in 70% ethanol and left to dry on the bench, for ~ 30 min, under a 60 W lamp, after which pellets were resuspended in 50-100 µl ddH2O. 100 µg of RNA was purified using a Qiagen RNeasy kit, using the specified protocol (RNeasy® Mini Handbook, Fourth Edition, April 2006).
Label
biotin
Label protocol
Biotinylated cRNA was prepared according to the standard Affymetrix protocol from 5 microg total RNA.
Hybridization protocol
Following fragmentation, 10 microg of cRNA were hybridized for 16 hr at 45C and at 60 rounds/min. The GeneChips were washed and stained in the Affymetrix Fluidics Station 400 and 450.
Scan protocol
GeneChips were scanned using the GeneChip Scanner 3000 7G.
