Total RNA was isolated just before processing for expression profiling. For preparation of total RNA individual organs were thawed in buffer containing chaotropic salt (RLT buffer, Qiagen) and homogenised using a Polytron homogeniser. Total RNA from individual samples was obtained according to manufacturer’s protocols using RNeasy Midi kits (Qiagen). The concentration was calculated from OD260/280 measurement and 2 µg RNA aliquots were run on a formaldehyde agarose gel to check for RNA integrity. The RNA was stored at -80°C in RNase free water (Qiagen).
Label
Cy3
Label protocol
For labeling 20 µg of total RNA were used for reverse transcription and indirectly labeled with Cy3 or Cy5 fluorescent dye according the TIGR protocol (Hedge et al., 2000). Labeled cDNA was dissolved in 30 µl hybridization buffer (6x SSC, 0.5% SDS, 5x Denhardt’s solution and 50% formamide) and mixed with 30 µl of reference cDNA solution (pool from six control animals) labeled with the second dye. HegdeP, Qi R, Abernathy R, Gay C, Dharap S, et al (2000): A concise guide to cDNA microarray analysis-II. Biotechniques 29: 548-562
Total RNA was isolated just before processing for expression profiling. For preparation of total RNA individual organs were thawed in buffer containing chaotropic salt (RLT buffer, Qiagen) and homogenised using a Polytron homogeniser. Total RNA from individual samples was obtained according to manufacturer’s protocols using RNeasy Midi kits (Qiagen). The concentration was calculated from OD260/280 measurement and 2 µg RNA aliquots were run on a formaldehyde agarose gel to check for RNA integrity. The RNA was stored at -80°C in RNase free water (Qiagen).
Label
Cy5
Label protocol
For labeling 20 µg of total RNA were used for reverse transcription and indirectly labeled with Cy3 or Cy5 fluorescent dye according the TIGR protocol (Hedge et al., 2000). Labeled cDNA was dissolved in 30 µl hybridization buffer (6x SSC, 0.5% SDS, 5x Denhardt’s solution and 50% formamide) and mixed with 30 µl of reference cDNA solution (pool from six control animals) labeled with the second dye. HegdeP, Qi R, Abernathy R, Gay C, Dharap S, et al (2000): A concise guide to cDNA microarray analysis-II. Biotechniques 29: 548-562
Hybridization protocol
This hybridization mixture was placed on a prehybridised microarray, under a cover slip, placed into a hybridization chamber (Genetix) and immersed in a thermostatic bath at 42°C for at least 16 hours. After hybridization slides were washed in 40ml of 3x , 1x, 0.25x and 0.1x SSC at room temperature. For drying slides were placed in an empty 50 ml Falcon tube (Becton Dickinson, USA) and centrifuged at 4000m/s2. Beckers, J., Herrmann, F., Rieger, S., Drobyshev, A., Horsch, M., Hrabé de Angelis, M. and Seliger, B. (2005): Identification and validation of novel ERBB2 (Her2, NEU) targets including genes involved in angiogenesis. Int. J. Cancer 114: 590-597. Greenwood AD, Horsch M, Stengel A, Vorberg I, Lutzny G, Maas E, Schädler S, Erfle V, Beckers J, Schätzl H and Leib-Mösch C (2005): Cell Line Dependent RNA Expression Profiles of Prion-infected Mouse Neuronal Cells. JMB 349: 487-500 Seltmann M, Horsch M, Drobyshev A, Chen Y, Hrabé de Angelis M, Beckers J (2005): Assessment of a Systematic Expression Profiling. Approach in ENU-Induced Mouse Mutant Lines. Mam Genome 16 (1): 1-10
Scan protocol
Dried slides were scanned with a GenePix 4000A microarray scanner and the images were analyzed using the GenePix Pro6.0 image processing software (Axon Instruments, USA) Greenwood AD, Horsch M, Stengel A, Vorberg I, Lutzny G, Maas E, Schädler S, Erfle V, Beckers J, Schätzl H and Leib-Mösch C (2005): Cell Line Dependent RNA Expression Profiles of Prion-infected Mouse Neuronal Cells. JMB 349: 487-500 Seltmann M, Horsch M, Drobyshev A, Chen Y, Hrabé de Angelis M, Beckers J (2005): Assessment of a Systematic Expression Profiling. Approach in ENU-Induced Mouse Mutant Lines. Mam Genome 16 (1): 1-10
Description
RNA isolation, Labelling, Hybridisation and Scanning procedure are described in the fields above
Data processing
Normalisation with GenePix Pro 6.0: Ratio based normalized data in each image so that the mean of ratio of means of all of the features is equal to 1. The VALUE column contains the log2 ratio of the result file after normalisation
