title: EMBL/CodeLink Rat 0.28K miCHIP v9.2
status: Public on Oct 06 2009
contact.city: Heidelberg
contact.first: Mirco
contact.last: Castoldi
contact.email: castoldi@embl.de
contact.address: Im Neuenheimer Feld 156
contact.department: Department of Pediatric Oncology, Hematology and Immunology
contact.country: Germany
contact.institute: University of Heidelberg
contact.phone: ++49(0)62213878410
organism: Rattus norvegicus
description: miChip array are spotted on CodeLink, GE Life Science, cat. no. 30001100). Codelink glass slides are coated with N-hydroxysuccinimide.
  
  NOTE: Probe sequences used in this array are proprietary, belonging to Exiqon.
manufacture_protocol: LNA-modified oligonucleotide (Exiqon) capture probes are designed to have a close to uniform Tm of 72 Celsius against their complementary targets. This is achieved by adjustment of LNA content and length of capture probes (miRCURY Array probes, Exiqon, http://www.exiqon.com). A C6-amino linker is added to the 5'-end of all capture probes. The dessicated capture probe set is available in a 384-well plate format. Capture probes are printed onto N-hydroxysuccinimide glass surfaces (Codelink, GE Healthcare Life Science) using the Lucidea Array Spotter (GE Healthcare Life Science).
  Additional material required for array preparation: post-coupling blocking solution [50 mM ethanolamine, 0.1 M Tris (pH 9)], post-coupling washing solution (4 XSSC, 0.1% SDS), a water bath (50 C) and a tabletop centrifuge (Eppendorf, 5810R) fitted with rotor (A4-62) adaptors for microplate carriers or for 50 ml tubes.
  miRCURY Array probes (Exiqon) are diluted to a final concentration of 20 uM in 3X printing buffer [(150 mM sodium phosphate (pH 8.5)] prior to spotting. Probe set are spotted on CodeLink, (GE Healthcare Life Science), where slides are printed using the Lucidea array spotter (GE Healthcare Life Science) with 65% humidity and a temperature of 21 C.
  Each spot measures about 120 um in diameter with a center-to-center spacing between spots of 300 um. To obtain a reliable read out each capture probe is printed in quadruplicate using a re-dip after four slides. Spotted glass slides are subjected to post processing as recommended by the manufacturer. In brief, CodeLink slides are designed to covalently immobilize amine-modified oligonucleotides for microarrays. However, the reactive groups are moisture sensitive and slides must be stored desiccated, hence the coupling of C6-amino linker oligonucleotides to the surface takes place in a humid environment following printing. Consequently, following printing, arrays are collected and introduced into bottom-sealed racks (e.g. microscope slide racks) that are placed in boxes containing a saturated solution of sodium chloride.
  (Note. To prepare a saturated solution of sodium chloride, add as much solid NaCl to water as needed to form a 1 cm deep slurry in the bottom of the box). Boxes are sealed with saran wrap or airtight lids, and stored overnight at room temperature. Next Day, printed arrays are transferred into glass racks and residual reactive groups on the CodeLink surface are inactivated by incubation with post-coupling blocking solution (pre-warmed to 50 C) for 30 min in the water bath (50 C). Then the slides are rinsed twice with deionized water and a third time with post-coupling washing solution (pre-warmed to 50 C) for 30 min on the shaker. Slides are then briefly rinsed with deionized water. Subsequently, the racks are placed into microplate carriers, and centrifuged for 2 min at 1500 rpm.
  Ready-to-hybridize slides can be stored for up to 6 month at room temperature in a dessicator.