title: Bos Taurus Expression Array for Smooth Muscle Gene Expression
status: Public on Feb 23 2006
contact.city: Boston
contact.first: Marc
contact.middle: E.
contact.last: Lenburg
contact.email: mlenburg@bu.edu
contact.state: MA
contact.address: 715 Albany Street, E613B
contact.department: Genetics and Genomics
contact.country: USA
contact.web_link: http://gg.bu.edu
contact.institute: Boston University School of Medicine
contact.phone: 617-414-1375
organism: Bos taurus
manufacture_protocol: Oligonucleotide Design:  50 mer oligonucleotides for 402 experimental genes related to smooth muscle function and 78 “house-keeping” genes were by first identifying 50bp sequences within the target cDNA sequence that were unique within the Bos taurus BTGI genome database (searches performed between January and February, 2004) and then filtering these to minimize predicted RNA secondary structure and to select sequences with a predicted annealing temperature of 84 +/- 6 degrees Celsius.  These steps were performed with the program ArrayOligoSelector (Bozdech Z, Zhu J, Joachimiak MP, Cohen FE, Pulliam B, DeRisi JL.  Expression profiling of the schizont and trophozoite stages of Plasmodium falciparum with a long-oligonucleotide microarray.  Genome Biology 2003; 4:R9.).  Among the five best candidate-probe sequences, the probe that hybridized to the most 3’ region of the target was selected.  For 54 genes that we wished to include on the microarray, an orthologous Bos taurus sequence had not been identified.  For these genes we searched the Bos taurus BTGI genome database for the most homologous sequence and used this in the probe selection process.
  
  Oligonucleotide Synthesis:  Oligonucleotides were synthesized on an ABI 3900 DNA Synthesizer (Applied Biosystems; Foster City, CA) on a 200 nmol scale using standard phorphoramidite chemistry. The 5’-DMT final group was retained for purification. All sequences were synthesized on 5’-DMT-mdC(TEG-FMOC) columns (Biosearch Technologies; Novato, CA) to provide an amino group for coupling to the array surface. Following synthesis, oligonucleotides were cleaved from the support and deprotected in conc. NH4OH at 65 °C for 5hr. The oligonucleotides were purified on Varian TOP columns (Varian Instruments; Walnut Creek, CA) following the manufacturer’s protocol. Quantitation was performed on a Wallac 1420 Victor¬2 plate reader (Perkin Elmer Life Sciences; Boston, MA) using an Oligreen ssDNA Quantitation Kit (Molecular Probes; Eugene, OR).
  
  Array Preparation: Oligonucleotides were aliquoted into 384-well spotting plates to produce a final concentration of 50 μM in 150 mM sodium phosphate pH 8.5. In addition to the gene specific oligonucleotides, the spotting plates contain SpotReport Alien Oligo sequences (Stratagene; La Jolla, CA) for use in balancing the intensities of the two signal channels and solvent blanks. Spotting was performed on a QArraymini (Genetix; Boston, MA) employing Stelth SMP4 pins (Telechem; Sunnyvale, CA). Oligonucleotides were spotted in duplicate onto Codelink slides (Amersham Biosciences; Piscataway, NJ) at 25 °C and 55% relative humidity, with the spotting plates chilled to 15 °C. Following spotting, the arrays were incubated for 24 hrs in a sealed container containing a NaCl slurry (~75% relative humidity). The arrays were washed, with agitation on a shaker, in 4X SSC; 0.1% SDS at 50 °C for 30 min, rinsed twice with water, and dried by centrifugation at 900 rpm for 10 min.