Dahlquist:DNA Microarray Protocol

Overview

This protocol describes all the steps necessary to perform aRNA synthesis, labeling, hybridization, and scanning of DNA microarrays for Saccharomyces cerevisiae, beginning with total RNA.

NOTE: This protocol is still under construction!

Materials

Supplies

NOTE: RNase-free supplies should be used whenever possible.

• SealRite 1.5 mL Natural Microcentrifuge Tubes (free of detectable RNase, DNase, DNA & pyrogens; USA Scientific Catalog #1615-5500)
• TipOne 101-1000 μL Filter Tips (sterile, free of detectable RNase, DNase, DNA & pyrogens; USA Scientific Catalog #1126-7810)
• TipOne 1-200 μL Graduated Filter Tips (sterile, free of detectable RNase, DNase, DNA & pyrogens; USA Scientific Catalog #1120-8810)
• TipOne 0.1-10 μL Filter Tips (sterile, free of detectable RNase, DNase, DNA & pyrogens; USA Scientific Catalog #1121-3810)
• 50 ml conical screw cap tubes, copolymer, bulk, sterile (USA Scientific Catalog #1500-1211)
• Powder-free latex exam gloves (USA Scientific Catalog #4900-2200, 4900-3300, 4900-4400)
• multiwipes, small and large (Denville Scientific Catalog #C5709-1A, C5709-3A)
  • a.k.a. Kim wipes
  • Denville Scientific sells a generic version that is less expensive than the name brand
• aluminum foil (from grocery store or other such supplier)
• compressed air duster
  • VWR sells something called the "Whoosh Duster" (VWR Catalog #16650-027), but I've found that one purchased from an electronics store (less expensive) works fine (Kam D. Dahlquist 20:46, 15 August 2008 (EDT))
• trUView™ Disposable Cuvettes (BioRad Catalog #1702510)
  • UV-transparant microcuvettes hold 50 μL total volume
  • can be cleaned and re-used indefinitely when you don't need to recover the sample from them
• RNaseZap (Denville Scientific Catalog #D1180)
  • Actually an Applied Biosystems/Ambion product Catalog #AM9780, but it is less expensive if you buy it from Denville

Reagents

NOTE: RNAse-free reagents should be used whenever possible.

• Amino Allyl MessageAmp™ II aRNA Amplification Kit (Applied Biosystems Catalog #AM1753)
  • 20 reactions per kit
  • Applied Biosystems Catalog Web Page. Originally an Ambion product. Ambion was acquired by Applied Biosystems and the kit can only be purchased through them.
  • Ambion Product Web Page. Contains useful links.
  • Ambion Protocol (PDF). Protocol packaged with kit.
• CyDye Post-Labeling Reactive Dye Pack (GE Healthcare Life Sciences Catalog #RPN5661)
  • 12 X Cy3 + 12 X Cy5 per box
  • GE Healthcare Product Web Page. Distributed through VWR.
  • Use protocol for Amino Allyl MessageAmp™ II aRNA Amplification Kit.
• TE Buffer (USB Catalog #75893)
• Nuclease-free water (non-DEPC treated)
  • Nuclease-free water is provided in the Amino Allyl MessageAmp™ II aRNA Amplification Kit and is sufficient for the μL quantities called for throughout the protocol; additional water can be purchased from Applied Biosystems/Ambion, but it is expensive.
  • DEPC is an oxidizer and should be fully removed from water used with DNA microarrays because the Cy5 dye is prone to degradation by oxidation.
  • For larger quantities of nuclease-free water (non-DEPC treated) that are needed to dilute 20X SSC for washes, for example, I use Water, ASTM Type II, non-sterile, Reagent Grade, ACS (VWR Catalog #RC91505) that can be purchased in 10 L or 20 L cubes. This water was recommended by Genisphere in the DyeSaver 2 protocol as being validated for use with microarrays and not containing components that will oxidize Cy5; Kam D. Dahlquist 20:24, 15 August 2008 (EDT))
• RNA Fragmentation Reagents (Applied Biosystems Catalog #AM8740)
  • Product information
  • 200 reactions per kit
• DIG Easy Hyb (Roche Applied Science Catalog #11796895001)
• Fish sperm DNA Solution 10 mg/mL
  • Have used Ultra Pure Salmon Sperm DNA Solution from Invitrogen (Catalog #15632-011)
  • When that runs out will use DNA, MB-grade from fish sperm from Roche Applied Science (Catalog #11467140001) which is significantly less expensive
• Oligo dA, 10-20mer, 1μg/mL (Invitrogen Catalog #POLYA.GF)
• 100% ethanol (Sigma Catalog #E702-3)
  • for adding to wash buffers in Amino Allyl MessageAmp™ II aRNA Amplification Kit
• 20X SSC (USB Catalog #19629)
  • for making post-hybridization wash buffers
• 20% SDS (USB Catalog #75832)
  • for making post-hybridization wash buffers
• Hybri-slips (Sigma Catalog #H0784)
  • pack of 100
  • inexpensive, disposable, easy-to-use
  • L × W × thickness 60 mm × 24 mm × 0.25 mm
• DNA microarrays
• Slide Coating Solution

Equipment

• Dedicated set of RNase-free pipetmen
• Speedvac
• Water bath set to 50°C
• Water bath set to 37°C
• Heat block set to 70°C
• Microcentrifuge
• Corning hybridization chambers
• racks (for 1.5 mL microcentrifuge tubes)
• racks (for 50 mL conical tubes)
• ice bucket and ice
• forceps
• slide box
• glass Coplin jar (to hold Slide Coating Solution)
• UV/vis spectrophotometer

Procedure

1. Step 1
2. Step 2
   • Step 2 has some additional information that goes with it.
3. Step 3
   1. Step 3 has multiple sub-steps within it.
   2. Enumerate each of those.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

1. List troubleshooting tips here.
2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
3. Anecdotal observations that might be of use to others can also be posted here.

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References

Relevant papers and books

If this protocol has papers or books associated with it, list those references here. See the OpenWetWare:Biblio page for more information.

Contact

• Who has experience with this protocol?
  • Kam D. Dahlquist
  • Genome Consortium for Active Teaching (GCAT)

or instead, discuss this protocol.