tissue: leaf cultivar: 'Cachac' treatment: control
All plant material used was obtained as sterile tissue culture from the Bioversity's International Transit Center (ITC) located in the Laboratory of Tropical Crop Improvement of Division of Crop Biotechnics, Katholieke Universiteit Leuven, Belgium.
Plants were grown at a density of 24 plants per table under a standardised light/dark regime of 12/12 h at 26/23oC respectively with a light intensity of around 10,000 lux (300 watts/m2) and a relative humidity of 75%. At the onset of the experiment, all plants were hand-watered until water drained freely from the base of the pots. The control plants were then watered once a day by flooding the tables with a fertigation solution and allowing the pots to stand for 30 min. Water-deficit (drought) conditions were imposed by withholding water supply for in total up to five weeks. Leaf samples consisted of a 5-8 cm wide strip (~3g fresh weight) removed from one side of the middle of the second, fully expanded leaf down from the top of the plant. Samples were snap frozen in liquid nitrogen and stored at -80oC until analysis. All sampling took place between 14:00 and 17:00 pm.
RNA was extracted using a modified Tris-LiCl method, based on the work of Tattersall et al. The modifications involved a DNAse treatment and an additional phenol:chloroform cleanup step. RNA concentrations and purity were determined using a MultiScan Spectrum microtitre plate scanning UV-VIS spectrophotometer (Multiskan Spectrum, Thermolabsystems, Brussels, Belgium). For purity assessment, the AU 260/280 and AU 260/230 ratios were measured and samples with a ratio of ~2.0 were considered as 'pure'. One microgram of total RNA sample was also run on a 1% agarose gel containing ethidium bromide in TAE buffer to check for possible degradation. Gels were imaged using a GelDoc 1000 gel imaging system and Molecular Analyst v1.5 image analysis software version 4 (Bio-Rad, Hercules, CA).
Approximately 5 ug of Musa total RNA was reverse transcribed at 42oC for 1 h to generate first strand cDNA using 100 pmol oligo dT(24) primer containing a 5'-T7 RNA polymerase promoter sequence, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol (DTT), 10 mM dNTPs and 200 units SuperScript II reverse transcriptase (Invitrogen Life Technologies). Following first strand cDNA synthesis, second strand cDNA was synthesised using 10 units of Escherichia coli polymerase I, 10 units of E. coli DNA ligase and 2 units of RNase H in a reaction containing 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)SO4, 0.15 mM beta-NAD+ and 10 mM dNTPs. This second strand synthesis reaction proceeded at 16oC for 2 h before 10 units of T4 DNA polymerase was added and the reaction allowed to proceed for a further 5 min. The reaction was terminated by adding 0.5 M EDTA. Double stranded cDNA products were purified using the GeneChip Sample Cleanup Module (Affymetrix). The synthesised cDNAs were transcribed in-vitro using T7 RNA polymerase (Enzo BioArray High Yield RNA Transcript Labelling Kit, Enzo Life Sciences Inc., Farmingdale, NY, USA) and biotinylated nucleotides to generate biotinylated complementary RNAs (cRNAs). The cRNAs were then purified using the Affymetrix Sample Cleanup Module (Affymetrix).
The cRNAs were randomly fragmented at 94oC for 35 min in a buffer containing 40 mM Tris-acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate to generate molecules of approximately 35 to 200 bp. Affymetrix A. thaliana ATH1 Genome or Rice Genome Arrays were then hybridised with 15 ug of fragmented, labeled cRNA for 16 h at 45C as described in the Affymetrix Technical Analysis Manual. GeneChip arrays were stained with Streptavidin-Phycoerythrin solution as described by the Affymetrix GeneChip Expression Analysis Technical Manual (http://www.affymetrix.com).
GeneChip arrays were scanned with an Affymetrix G2500A GeneArray scanner, as described in the Affymetrix GeneChip Expression Analysis Technical Manual.
Control RNA from leaves of the Musa cultivar 'Cachac' onto Rice array.
The Microarray Analysis Suite (MAS Version 5.0; Affymetrix) was used to generate .cel files for each of the RNA hybridisations by scanning and computing summary intensities for each probe without the use of probe mask files. These .cel files were then loaded into the GeneSpring (Agilent Technologies) analysis software package using the Robust Multichip Average (RMA) pre-normalisation algorithm. During .cel file loading and pre-normalisation, .cel files were interpreted using the respective A. thaliana or rice .cdf files (i.e. with no probe-selection used), or (2) using .cdf files generated from the Musa genomic DNA .cel file with CDF values ranging from 0 to 1000. Following RMA pre-normalisation and masking of individual probes, each probeset signal value from treated (drought stressed) samples was standardised relative to the probeset signal value of its corresponding control, to give the relative gene expression ratios between the two conditions.