Experimental Design.
Michael G Salter, Keara Franklin and Garry C Whitelam.
Biology Department, Adrian Building, The University of Leicester, University Road,
Leicester LE1 7RH.
Correspondence to mgs5@le.ac.uk
Type of experiment.
This experiment was a time course performed over 24 hours to look at the effects on
gene expression of exposure to low red:far-red ratio light in Arabidopsis thaliana
plants. In this way genes involved in the shade avoidance response might be
identified.
Experimental Factors.
The sole purpose of the experiment was to identify previously unreported genes which
respond to low R:FR light. For this reason no replicate arrays were performed though
the 24 h time point may be perceived to be a replicate of the 1 h time point. Seedlings,
ecotype La-er grown under 8 h light/16 h dark cycles until the 11-leaf stage were
exposed to supplementary far-red light beginning 1 h after dawn. Tissue was
harvested for RNA extraction at 1 h and at 8 h after the onset of supplementary far-red
light (daylength extended by 1 h). Tissue was also harvested again after a further 1 h
of low R:FR ratio commencing immediately with the following dawn. At all time
points samples were taken from treated and untreated plants. Each sample consisted
of an amalgam of the aerial parts of six individuals. Plants maintained in high R:FR
ratio white light received a photon irradiance of 400-700 nm at 130 µmol m-2 s-1 and a
R: FR ratio of 4.7. The supplementary far-red treatments received the same photon
irradiance, but a R:FR ratio of 0.089.
Hybridisation Strategy.
A total of 7 arrays were used, one array per treatment. The array used was the
Affymetrix 8200 Arabidopsis array, part number 510429, lot number 9916505. The
experiment was designed to enable comparison between treated and untreated groups
at specific time points as well as backwards comparison of all samples with the T0
sample.
Extraction Procedures and Sample Preparation.
Samples consisted of predominantly leaf tissue and RNA was extracted using
QIAGEN RNeasy midi kits in accordance with the manufacturer’s instructions.
Following extraction the RNA was subjected to extraction with acid phenol followed
by extraction with chloroform: isoamyl alcohol 24:1 and ethanol precipitation to a
final concentration > 3 mg/ml. Sample tubes were marked with time point and either
W for high R:FR ratio control samples and FR for low R:FR ratio-treated samples.
Labelling for hybridisation was completed using Enzo Life Sciences BioArray™
HighYield™ RNA Transcript Labeling kit. Biotin-labeled RNA is produced by T7
RNA polymerase-catalyzed in vitro transcription in the presence of biotin-labeled
UTP and CTP. The biotin labelled cRNA is fragmented and hybridised to the
Arabidopsis array and stained with phycoerythrin-streptavidin and scanned to
generate the raw image dat file.
Measurement of Data and Analysis.
Scanning was completed using the full Affymetrix Gene Chip® instrument system.
Data from the Gene Chip instrument system was analysed using the Gene Chip®
analysis software. Quality control data for individual chips supplied as separate text
files.
To date no cluster analysis has been performed on these data. Data from the Gene
Chip analysis system was ordered within Excel files to identify genes by fold
expression change. Analysis consisted of selecting the ten most highly regulated
genes for each time point, treated vs untreated, and referral back to the genetic
databases for confirmation of potential protein predictions and annotation.
Array Design.
Array design was as specified by Affymetrix, information available at
http://www.affymetrix.com.
Sample Identification.
T0. Sample was taken when plants were moved from high R:FR white light to low
R:FR ratio (white plus supplementary far-red light) 1 hour after lights on.
F1. Sample was taken from plants which had been treated with low R:FR ratio for 1
hour
W1. Sample taken from plants maintained in high R:FR ratio white light 1 hour after
T0 sample.
F8. Sample was taken from plants treated with low R:FR ratio light for 8 hours
continuously.
W8. Sample was taken from plants maintained in high R:FR ratio white light for 8
hours after T0.
Following F8/W8 samples plants were moved into darkness for 15 hours.
F24. Sample was taken from plants given 1 hour of low R:FR ratio commencing at
lights on. These plants had also received 8 hours of low R:FR ratio light during the
previous day
W24. Sample was taken from plants which had been maintained in high R:FR ratio
white light for 1 hour after lights on.
Quality Control Data
See quality control spreadsheet for each individual array.