treatment: Alethea pre-treatment, salt main treatment cultivar: Ailsa Craig tissue: Leaf age: 4 weeks
Treatment protocol
An experimental formula of the ‘Alethea’ technology (Plant Impact PLC, Harpenden, UK) was applied to four-week old tomato seedlings at a concentration of 99:1 v/v (distilledH2O:Alethea) as per manufacturer’s instructions. Control plants, were sprayed with distilled H2O. Alethea solution was sprayed onto leaves until run-off using a pressurized airbrush. Plants were air-dried and then returned to the glasshouse. 24 h following Alethea application, a salinity treatment of 100 mM NaCl was applied to plants via pot-watering until maximum soil saturation was reached (~ 3 h), with control plants fed with H2O only. 24 h following salinity treatment (48 h following Alethea treatment), 3rd true leaves of plants were snap frozen in liquid nitrogen for subsequent RNA extraction.
Growth protocol
Tomato seed (cv. Ailsa Craig, Moles Seeds Ltd, Colchester, UK) were sown and germinated in Levington M3 compost (Henry Alty Ltd., Preston, UK), prior to individual transplantation into 2 L pots. Seedlings were maintained in glasshouse conditions supplemented with high pressure sodium lighting, supplying a background Photosynthetically Active Radiation (PAR) photon flux density of approximately μmol m-2 s-1, with a photoperiod of 14 h/10 h light/dark, and air temperatures of 22°C/18°C day/night. Plants were arranged randomly according to treatment, and were grown for four weeks prior to establishment of experimental treatments (approximately 6th true leaf stage).
Extracted molecule
total RNA
Extraction protocol
For each treatment, a single RNA extract was made from leaf 3 pooled from 3 individual plants. 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
Labelled using protocol described in Affymetrix manual 3' IVT Express Kit User Manual
Hybridization protocol
Biotin Labelled cRNA using Affymetrix GeneChip Hybridization, Wash and Stain Kit, according to guidelines set out by Affymetrix, Santa Clara, CA
Scan protocol
Scanned according to guidelines set out by Affymetrix, Santa Clara, CA.
Data processing
Probe signal intensities were extracted from .CEL files and normalised using the GC-RMA method (default settings) in the R Bioconductor suite.
