Briefly, 20 part normalized cDNA libraries were prepared from 3 28 DAP endosperm and kernel development tissues covering the 5 key stages software. For each contig, the cDNA contain ing the largest transcript was identified. These, together with all singleton cDNAs were used to construct a Unigene set of 8,950 sequences. ESTs were stored as cloned fragments in glycerol stocks in 384 well microti ter plates at 80 C. Before spotting, 2 ul of each EST sample were added to 50 ul PCR amplifications using, 2 ul of T3 primer at 15 pmol ul, 2 ul of T7 primer at 15 pmol ul, 5 ul of 2 mM dNTP mix, 1. 5 ul of 50 mM MgCl2, 5 ul of Invitrogen 10x PCR reaction buffer, 0. 2 ul of Invitrogen Taq DNA polymerase recombinant. Amplified products were purified with the Wizard MagneSil PCR Clean Up System.

Aliquots were then tested on 0. 8% agar ose gels in order to verify insert integrity and concentra tion. Finally, selected amplification products were air dried and resuspended in 15 ul of 3x printing buffer. mRNA isolation and slide hybridization Inhibitors,Modulators,Libraries Total RNA was prepared from 100g frozen, ground endosperm tissue using Inhibitors,Modulators,Libraries Trizol Reagent following the manufacturers instructions. polyA RNA was purified using the PolyA Tract mRNA System Kit IV following two cycles of oligo column purification to ensure a high purity of polyA RNA. The purified RNA was quantified Anacetrapib by measuring its absorbance at 260 nm and diluted to a final concentration of 1 ug ul. For each mRNA probe, 1 ug of purified polyA RNA was labelled by reverse transcription in the presence of Cy3 and Cy5 dCTP using the Amersham CyScribe First Strand cDNA Labelling kit following manufacturers indications.

Microarray slides were placed in a rack and incubated as follows, 1 15 30 min at 50 C in pre warmed pre hybridization solution 1, 2 two rapid Inhibitors,Modulators,Libraries washes in distilled water, 3 20 40 min at 50 C in pre warmed pre hybridization solution 2, 4 2 min in distilled water at 94 C for sample denaturation, 5 two washes at RT in distilled water. Subsequently, slides were centrifuged at 1,500 rpm for 3 min at RT. Labelled cDNA mixes were added to 15 ul of formamide and 7. 5 ul of Amersham 5x microarray hybridization buffer. The mixture was dis pensed onto the microarray slides, covered with a Hybri Slip cover slip and incubated in the dark at 42 C overnight in a hybridization chamber containing 120 ul of sterile distilled water to maintain humidity.

Hybridized slides were washed as follows, 1 5 min at 42 C with 2x SSC, 0. 1% SDS, 2 5 min at 42 C with 1x SSC, 0. 1% SDS, 3 5 min at RT in 0. 2x SSC, 4 5 min at RT in 0. 1x SSC, 5 5 min at RT in distilled water. Finally, the slides were centrifuged at 1,500 rpm for 3 min to remove Inhibitors,Modulators,Libraries remaining liquid. Microarray data analysis All microarray experiments were performed in triplicate using dye swapping, hence giving rise to 12 independent measurements for each EST, considering the presence of duplicate spots on each slide.