Ferreira et al. BMC Genomics 2010, 11:93 http://www.biomedcentral.com/1471-2164/11/93 qPCR analysis For all qPCR analysis at least three biological repeats were used unless stated otherwise. Expression levels of genes were determined by real-time quantitative RT- PCR and the corresponding primers for the amplifica- tion of targets between 75 and 150 bp were designed using Primer3plus software [55]. Total RNA (five μg) from each of the developmental time points was treated with DNaseI (Fermentas GmbH) before undergoing reverse transcription using oligo d(T) primers and RevertAid™ H minus first strand cDNA synthesis kit (Fermentas GmbH) to generate a first strand cDNA template. Potato ubiquitin primers were used as a con- trol as described previously [26]. One μl of 1:10 diluted cDNA for each time point were amplified with gene- specific primers in three technical replicates on a Mx3000P Q-PCR system (Stratagene) in combination with the Brilliant II SYBR Green Q-PCR Master Mix Kit (Stratagene). The thermal profile was as follows: 1 cycle 10 min at 95°C for DNA polymerase activation followed by 35 cycles of 10 s at 95°C, 15 s 60°C and 20 s 72°C. The primer sequences were as follows: ubi3 (L22576) forward primer, 5’-TTCCGACACCAT CGACAATGT- 3’; reverse primer, 5’-CGACCATCCTCAAGCTGCTT- 3’. For GBSS the primer sequence was based on POCI feature micro.920.c2. The forward primer was desig- nated GBSS_920.c2 F (5’ - CAGACTTGAGGAGCA- GAAAGG - 3’) and the reverse primer GBSS_920.c2 R (5’ - GTGAGCCAAAGGGACATTGA - 3’). For GPT the primer sequence was based on POCI feature micro.1076.c1. The forward primer was designated GPT_1076.c1 F (5’ -CCTTGTTTCCTGTTGCTGTG- 3’) and the reverse primer GBSS_1076.c1 R (5’ -AAAG- CAGGCTCTCCACTCTT- 3’). For Susy the primer sequence was based on POCI feature micro.196.c8. The forward primer was designated Susy_196.c8 F (5’ -CTGCTGTTTATGGGTTCTGG- 3’) and the reverse primer Susy_196.c1 R (5’ -GGCACACCTTCATT- CACTCA- 3’). For NTT the primer sequence was based on POCI feature micro.1831.c2. The forward primer was designated NTT_1831.c2 F (5’ -GAGCAGCAGCCAA- GATAACAC- 3’) and the reverse primer GBSS_1831.c2 R (5’ -GTTCTGCATTGCACCCACA- 3’). Relative gene expression was calculated using the Pfaffl method [56]. Description of X-ray CT With X-ray CT the 3D volume information of objects can be reconstructed using X-ray projections of the object from different aspects. The geometry used for the investigation was the axial 3D-CT, where a conical X- ray beam projects the object onto a flat 2D image detec- tor. Using axial 3D-CT, projections of the object are taken under different viewing angles, rotating the object perpendicular to the central X-ray beam. The recon- structed volume data set consists of volumetric Page 15 of 17 elements, called voxels, containing grey levels which represent information about the X-ray attenuation char- acteristics depending on the mass attenuation coefficient and the density distribution of the material [57,58]. The mass attenuation coefficient itself is dependent on the applied X-ray spectrum and the effective atomic number of the X-rayed material. The calculation of tuber volumes of potted potato plants embedded in soil requires the segmentation of tubers from other materials in the X-ray CT volume data. Therefore a careful selec- tion of exposure conditions is necessary to achieve suffi- cient data quality. This comprises X-ray parameters and filters as well as the condition of the soil. The para- meters were defined as such: The X-ray source was FXE-225.45, accelerating voltage 200 kV, total emission 200 μA, the filter 1 mm Cu, detector Perkin Elmer, 1024 × 1024 pixel 200 μm, scan period 50 minutes and a resolution of 141 μm. After segmentation voxel ele- ments, which have a known volume and specific grey level for tubers, were used to determine tuber volume by calculating how many voxels are present in a recon- structed tuber image. The lower and upper threshold grey values were set at 2938 and 3963 after which mis- takes were corrected for manually. Tuber volume calcu- lations were performed using Image J software http:// rsbweb.nih.gov/ij. Acknowledgements The authors would like to acknowledge Christine Hösl for plant care. The work was funded by the BMBF in frame of the GABI-FUTURE program. Author details 1Friedrich-Alexander-University Erlangen-Nuremberg, Department of Biology, Staudtstrasse 5, 91058 Erlangen, Germany. 2Fraunhofer Institute for Integrated Circuits IIS, Development Centre X-Ray Technology, Dr-Mack- Straße 81, 90762 Fürth, Germany. 3Fraunhofer Institute for Integrated Circuits IIS, Contactless Test and Measuring Systems, Am Wolfsmantel 33, 91058 Erlangen, Germany. Authors’ contributions SJF did most of the experimental work and also drafted the manuscript. MS helped with the tuber volume calculations, microarray data analysis and functional assignment of features. SS helped with microarray hybridisations and data analysis. PK supervised and RG initiated the X-ray CT work. US provided the project funding, conceived and led the study and significantly contributed to drafting the manuscript. All authors read and approved the final manuscript. Received: 7 August 2009 Accepted: 5 February 2010 Published: 5 February 2010 References 1. 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