title: National DNA microarray facility of University of Aveiro miRNA chip v1.1
status: Public on Jul 27 2009
contact.city: Aveiro
contact.first: Patrícia
contact.middle: Matos
contact.last: Pereira
contact.email: pereirap@ua.pt
contact.address: Campus Universitário de Santiago
contact.department: Biology
contact.country: Portugal
contact.institute: University of Aveiro
organism: Homo sapiens
  Mus musculus
  Rattus norvegicus
manufacture_protocol: miRNA chip v1.1 CONTENTS:
  The spotted oligo library was mirVana miRNA probe set V1.0 from Ambion. This library contains 382 42-46mer amine-modified DNA oligonucleotides targeting 281 human, 14 rat and 49 mouse mature miRNA sequences from miRBase Sequence Database version 7.1. In addition, the mirVana miRNA Probe Set includes 33 probes targeting an exclusive set of newly identified human miRNAs, called Ambi-miR™ miRNAs and one positive control and 4 negative controls. The library is provided in 964-well plates in lyophilized form.
  The array also included a set of 4 hybridization controls (External Controls) for the detection of RNA spike mixes added to RNA extracts during sample preparation. The control probes were designed so that they had no homology with human, mouse and rat genome and matched in Tm and GC content with mirVana miRNA probe set.
  LIQUID HANDLING: The goal was 20 uM oligos in 3XSSC buffer in 384-well plates (Genetix X-6004) ready for robotic spotting.
  1. Lyophilized oligos (500 pmol in 96-well plates) were resuspended in 3XSSC to a concentration of 20 uM using a liquid handling station (Sciclone LH 3000, Caliper).
  2. After shaking overnight at 4 ºC, at 100 rpm, part of each dissolved oligo was transferred to 384-well plates. The 384-well plate was stored at -80 ºC ready for spotting.
  3. The remaining stock in 384-well plates was air dried at room temperature and stored at -80 ºC. When necessary, milliQ water was used to resuspend the oligos accounting for lost volume.
  4. The spotting probe set also included 4 External Control (EC) oligos (44mer, lyophilized, provided in separate microfuge tubes). These oligos were resuspended in milliQ water to a concentration of 100 uM. Aliquots for spotting were diluted to a final concentration of 20 uM oligo in 3XSSC. Remaining 100 mM stocks were stored at -20 ºC. Spotting aliquots were air dried at room temperature and stored at -80 ºC, together with the Ambion miRNA probe set.
  SPOTTING:
  1. The 42-46 mer probes were transfered from the 384-well plates and printed on Nexterion E slides (SCHOTT) with a BioRobotics MicroGrid Compact II spotter (Genomic Solutions) in an environment controlled room. Humidity was 60% ± 2%. Temperature was 21 ºC in the spotter. Quill Pins type MicroSpot2500 (BioRobotics) were used for spotting.
  2. For probe fixation, the printed slides were incubated in a humidity chamber at room temperature, overnight.
  3. Slides were stored in a desiccator-cabinet (Nalgene, with blue Silica (Sigma)) ready to use (continue with our hybridization protocol).