Ferreira et al. BMC Genomics 2010, 11:93 http://www.biomedcentral.com/1471-2164/11/93 Page 14 of 17 Figure 14 Expression profiles of transcription factors possibly regulating starch biosynthesis. A) Diurnally in leaves, B) during tuber induction and C) growing and non-growing tubers. Micro.8007 (AT2g40820), orange, Micro.7865.c1 (AT3G16280), blue, Micro.5635.c1 (AT4G37750), black, Micro.5579.c2 (AT4G34590), light blue, Micro.15471.c1 (AT4G00870), brown and Micro.4326.c1 (AT4G32730), red. Technologies). To check for RNA degradation two μg of total RNA were separated on 1.5% formaldehyde con- taining agarose gel. Total RNA was purified using RNeasy Mini Spin Columns (QIAGEN, Valencia, CA). Afterwards RNA quality and quantity was tested using the Agilent 2100 BioAnalyzer (vB.02.03 BSI307) as recommended by manufacturer’s protocol (Agilent RNA 6000 Nano Assay Protocol2). Synthesis of cDNA and cRNA was performed as described in the one-color microarray-based gene expression analysis protocol pro- vided by Agilent including the one-color RNA spike-in kit (v5.0.1, 2006; Agilent Technologies, Santa Clara). After fragmentation Cy3-labelled samples were loaded on the array and hybridised over night (17h/65°C). Slides were washed as recommended in the manufac- turer’s protocol and scanned on the Agilent Microarray Scanner with extended dynamic range (XDR) at high resolution (5 μm). Data sets were extracted by using the feature extraction software (v9.5.3.1/Agilent Technolo- gies) using a standard protocol. Array data analysis Data were imported into GeneSpring GX 7.3.1 (Silicon Genetics, Palo Alto, CA, USA) and additionally stored on a local server. A three step normalization was applied: (1) values less than five were set to five, (2) per chip normalization to 50th percentile and (3) subse- quently the signal for each feature was normalised to the median of its value across the entire dataset. For comparative analysis of various transcription profiles, a volcano plot was applied to select for features more than two-fold differentially expressed between two con- ditions including the Benjamini-Hochberg multiple test correction for the four replicate experiment comparing slow and fast growing tubers. No multiple test correc- tion was employed for the experiments comparing sto- lon and swollen stolon, and the diurnal leaf experiment, where only two replicates per data-point were used. Fea- tures commonly differentially expressed in all experi- ments were identified using a Venn diagram. K-means clustering was performed using Pearson correlation to split the selected features into five clusters from where features were chosen to conduct a functional assignment. Starch and sucrose measurements Sucrose and starch were measured according to a modi- fied method of Müller-Röber et al [17]. To determine sucrose supply rate to a tuber, sucrose content of sto- lon-ends was determined. A stolon-end is defined as the 15 millimetres of a stolon directly above the connection to the tuber.