The Huck Institutes of the Life Sciences

NimbleGen microarray

Protocols for NimbleGen 4-plex and 12-plex microarrays

  1. Accurately determine RNA concentration and purity using NanoDrop and Qubit if necessary.
  2. Assess total RNA quality using RNA Nano Chips on the Agilent Bioanalyzer. RINs are generally 7-10 but consistency and range of scores is most important.
  3. Amplify 1 ug of each sample using the Ambion (Life Technologies) Amino Allyl MessageAmp II aRNA Amplification Kit (AM1753). Follow the manufacturer’s protocol for one cycle amplification.  Normal aRNA yield is approximately 60 ug with a range of 30-100 ug.
  4. Dye couple 15 ug of aRNA with Cy3 or Cy5 (GE Health Care #RPN5661), as appropriate, and subsequently purify according to the Ambion Kit instructions. A 10x dilution should have approximately 60 ng/ul of material with a range of 50-100 ng/ul.
  5. For both 4-plex and 12-plex arrays 1.5 ug of a Cy3 labeled sample is combined with 1.5 ug of a Cy5 labeled sample and fragmented using RNA Fragmentation Reagents (Ambion AM8740) according the manufacturer’s instructions.
  6. Following fragmentation, dry samples down completely in a speed-vac.
  7. Resuspend each pair of samples in the appropriate Tracking Control and add Hybridization Solution Master Mix according to manufacturer’s instructions. (Roche NimbleGen).
  8. Incubate at 95 C for 5 minutes, followed by 42 C for 5 minutes. Attach appropriate mixer to the array and add hybridizations solution according to the manufacturer’s instructions. Hybridize with mixing in a MAUI hybridization instrument overnight at 42 C.
  9. Disassemble and wash arrays according to the manufacturer’s instructions (Roche NimbleGen).
  10. Spin at 1000 RPM for 3 min. in a 50 ml conical tube previously filled with N2 gas.
  11. Make sure slide is completely dry. Allow to air dry momentarily remove any excess buffer from slide edges with a Kimwipe.
  12. Place slide in a fresh tube and add 2.5 ml of Dye Saver. (Genisphere Inc). Coat the slide with DyeSaver by rotating the tube manually for 45 seconds.
  13. Spin at 700 RPM for 3 seconds down to remove excess DyeSaver.
  14. Remove from tube and allow to air dry briefly in a hood.
  15. Scan using Axon GenePix 4000B.