Experimental design. A. B stands and M protoplasts were isolated by mechanical disruption and enzymatic digestion, respectively. Representative chloroplasts are marked with an arrow. B. Interwoven loop design of microarray labelling. Four treatments (B, M, S, T) were compared using two-dye microarrays. Each arrow represents hybridization. RNA isolated from the treatment at the tail of the arrow was used to synthesize Cy3-labelled cDNA, and RNA isolated from the treatment at the head of the arrow was used to synthesize Cy5-labelled cDNA. Independent biological replicates were used for each labelling, giving a total of six per treatment. Numbers on arrows refer to Additional file 1. C. Pseudo-colour overlay of Cy3 and Cy5 images from a representative hybridization. A single 26 × 26 features sub-grid is shown. The complete University of Arizona oligonucleotide array consists of 96 such grids arrayed over two slides.