title: RetroArray - a DNA chip for rapid detection and identification of retroviruses
status: Public on Jun 10 2011
contact.city: Neuherberg
contact.first: Michelle
contact.last: Vincendeau
contact.email: michelle.vincendeau@helmholtz-muenchen.de
contact.address: Ingolstädter Landstr. 1
contact.country: Germany
contact.institute: Helmholtz Zentrum München
organism: Macaca mulatta
  Homo sapiens
manufacture_protocol: For preparation of DNA chips, probably any standard robotic device will provide satisfactory results. We employ an Affymetrix GMS 417 Arrayer (Affymetrix, Santa Clara, CA, USA). Corresponding pairs of 45-mer synthetic oligonucleotides are diluted to 100 mM in 3x SSC (1x SSC = 0.15 M NaCl plus 0.015 M sodium citrate) and mixed in equimolar amounts. Capture probes are spotted (three hits per dot) onto CSS-100 aldehyde-activated glass slides (TeleChem International Inc., Sunnyvale, CA, USA). Generally, to rule out local defects in the slide surface (e.g. scratches) and for hybridization reproducibility, two replicas are spotted onto the same slide. After spotting, slides are incubated in a humid chamber over night, then air dried, washed and treated with sodium borohydride (NaBH4) as recommended by the glass slide manufacturer. Slides are stored in the dark at room temperature until hybridization. For the standardized experimental flow see Seifarth et al., 2003.
  Reverse transcription of RNA was performed using 1 μg total RNA with Superscript II (Roche Diagnostics) according to the manufacturer’s protocol. Reverse transcriptions were followed by an RNase H digestion step (Promega).
  RetroArray hybridization is carried out according to a standardized protocol as initially described by Seifarth and coworkers [2003]. It includes the following steps:
  1) Prehybridization 2) Hybridization 3) Washing 4) Drying of hybridized glass slides
  In general, all handling steps involving fluorochromes are performed under dimmed light conditions, incubations are carried out in the dark. Prehybridization of glass microarrays is performed within Falcon blue cap tubes (Greiner GmbH, Nürtingen, Germany) in 6x SSC, 0.5% sodium dodecyl sulfate (SDS), 1% bovine serum albumin (DNase-free) at 50 °C for at least 4 h. Slides are then washed five times (1 min each) in bidest. water under vigorous agitation. Subsequently, slides are dried by a short centrifugation step (500x g for 1 min at 22 °C). For hybridization, a 22x22 mm glass lifter slip (Erie Scientific Co., Portsmouth, NH, USA) is taped to the microarray. Subsequently, 25 μl of the denatured (5 min at 95 °C) Cy3-labeled probe (containing 3x SSC, 0.1x SDS, 50% formamide) is applied to the small space between cover slip and microarray and sealed with Fixogum rubber cement (Marabu GmbH, Tamm, Germany). Sealed slides are incubated in a humid chamber for at least 16 h at 42 °C. Hybridized slides are then washed three times under vigorous agitation (10 min each) at 22 °C in 1x SSC, 0.5x SSC and 0.1x SSC, respectively. After a brief centrifugation step (500x g for 1 min at 22 °C), the hybridized microarrays were stored in the dark at 22 °C until evaluation by scanning.
  Hybridized microarrays were scanned using an Affymetrix GMS 418 scanner (laser power setting, 100%; gain setting, 50%), and the resulting images (16-bit TIFF files) were subjected to densitometric analysis using ImaGene 4.0 software (BioDiscovery Inc.). To discriminate positive signals from background, a cut-off value of 1,200 relative fluorescence intensity units was used that corresponded to twofold overall background intensity value of the microarray. To account for the influence of RNA quality, HERV signals were normalized to the fluorescence intensity of a House keeping gene.