title: RASTA Rattus norvegicus 0.3 k ver. 1
status: Public on Aug 01 2009
contact.city: Berlin
contact.first: Oleg
contact.last: Tchernitsa
contact.email: olegq@hotmail.com
contact.address: Chariteplatz 1
contact.department: Institute of Pathology
contact.country: Germany
contact.web_link: http://mtp.charite.de
contact.institute: Charite
contact.phone: ++49 30 450536107
organism: Rattus norvegicus
manufacture_protocol: The Ras signaling target array was designed and produced as described
  (Tchernitsa et al., 2005). Briefly, we selected 121 genes recovered from subtracted cDNA libraries representing sequences differentially expressed in parental and KRAStransformed ROSE 199 cells (Tchernitsa et al., 2004). In addition, we selected genes from subtracted libraries representing sequences differentially expressed in normal 208F
  fibroblasts and the HRAS-transformed derivative FE-8 (Zuber et al., 2000). Previously described Ras pathway targets and 13 different genes described as housekeeping 20 genes in many different tissues and cell lines were included to permit normalization of hybridization intensities on microarrays. The total number of gene probes is 324 on the
  array.
  Unmodified 70-mer DNA oligonucleotides were chosen as probe sets for the array. The oligonucleotide design was performed with software available on the web (http://oligo.lnatools.com/expression/) using the following parameters: option for LNA frequency=0, melting temperature between 74캜 and 76캜, GC content between 45-55%, absence of strong secondary structures and proximity to 3’ end of coding sequence. Oligonucleotides were synthesized at Illumina, Inc (San-Diego, USA) at the 50 nmol scale. Super Frost glass slides (Menzel GmbH, Braunschweig, Germany) were soaked in 200 ml of washing solution containing 80 ml of 5M NaOH and 120 ml of 95%
  ethanol and agitated by a magnet stirrer for 5-6 h. Slides were washed 10-times with 200ml of ddH2O, incubated for 30 min on a shaker with poly-L-lysine solution (20ml of poly-L-lysine, Sigma-Aldrich, St. Louis, USA, mixed with 160 ml of ddH2O and filtered) and again washed 10-times with 200 ml of ddH2O. Afterwards, the remaining washing
  solution was removed by centrifugation of slides in a microarray centrifuge (Telechem, Sunnyvale, USA) and the slides were dried in an incubator for 2 h at 45oC. The oligonucleotides were adjusted to a concentration of 20킡 in 3xSSC/0.01% SDS buffer and spotted using a MicroGrid compact microarrayer (Genomic Solutions, Ann Arbor, USA) equipped with MicroSpot 2500 quill pins. The pins have a diameter of
  100 탆 and typically deliver spot diameters of 180 탆. Microarrays were printed at a relative humidity of 50 - 60%. Pins were rinsed twice in distilled water between each probe aspiration/dispense cycle. Each of the 325 oligonucleotides was spotted four times at a different location on the slides. Thus, the array contains 1,300 features. After printing, microarrays were re-hydrated twice by placing them over boiling water for five sec each. Then each array was snap-dried on a hot plate (90캜) for 10 sec (DNA side 21facing upwards). Slides were cross-linked with an intensity of 60mJ (UVC-500 crosslinker, Hoefer, Inc, USA). Afterwards, slides were incubated in blocking solution (3.2 g of succinic anhydride mixed with 200ml of N-methyl-pyrolidone and 4.45 ml of 1M sodium borate) for 20 min on an orbital shaker, rinsed three-times with ddH2O and dried by centrifugation.