Skip to Content

Using Perkin Elmer Lifesciences Micromax Direct Labeling Kit

General Instructions

Overview of Methodology (Direct Labeling) [link ppt file]

  • The following protocol is an abbreviated version of the protocol that accompanies the kit. Read through the kit protocol before following the steps below
  • Use only RNase/DNase free reagents/supplies. Wear gloves at all times
  • Cy3/Cy5 dyes and reagents containing these dyes are light sensitive. Care should be taken to minimize exposure of these reagents to light

Determination of Labels and Amount of RNA Needed

  • The untreated/control sample should be labeled with Cyanine 3 (Cy3)
  • The treated sample should be labeled with Cyanine 5 (Cy5)
  • 50 µg of total RNA is required per sample

cDNA Synthesis

  1. Add dNTP/Primer Mix (tube X), unlabeled control RNA (tube W), if needed, total RNA and water to a 1.5-mL Eppendorf tube labeled Cy3 and Cy5, according to the following table:

    One set of reactions

    ReagentsCy3 (µL)Cy5 (µL)
    dNTP/Primer Mix (tube X)2.02.0
    Unlabeled Control RNA (tube W)*2.02.0
    Total RNA ** (y)  
    Water ***  
    Total17.019.0

  2. Incubate at 65° C for 10 minutes, then at 25° C for 5 minutes. 
  3. Add Cy dyes: 4 µL of Cy3 dye to each Cy3 tube and 2 µL of Cy5 dye to each Cy5 tube.
  4. Incubate at 42°C for 2-3 minutes and add to each tube the following:

    2.5 µL RT Reaction Buffer (Tube A)
    2.0 µL AMV RT/RNase Inhibitor Mix (Tube E)
  5. Incubate at 42°C for 60 minutes. Then, 4°C for 10 minutes. Add to each tube the following:

    2.5 µL of 0.5 M EDTA (Mix well)
    2.5 µL of 1.0 N NaOH (Mix well by vortexing)

    Then, spin down at max speed for a few seconds. 
  6. Incubate at 65°C for 30 minutes. Then, 4°C for 5 minutes, during which add 6.2 µL of 1M Tris HCl, pH 7.5, to each tube. 
  7. Combine the Cy3 and Cy5 tubes for each hybridization reaction.

cDNA Purification

Use Millipore Microcon YM-100 Column (Catalog No. 42413)

  1. Place membrane cartridge in a clean 1.5 mL eppendorf tube.
  2. Add 200 mL of 10 mM Tris, pH 7.5 to membrane cartridge. Add Cy3 and Cy5 reactions to the membrane cartridge.
  3. Centrifuge the sample at 500 x g for 4 to 6 minutes, until sample volume reaches about 20 mL.
  4. Add 500 mL of 10 mM Tris, pH 7.5 to membrane.
  5. Centrifuge the sample at 500 x g for 4 to 6 minutes, until sample volume reaches about 20 mL.
  6. Invert cartridge into clean 1.5 mL tube and centrifuge at 500 x g for 4 to 6 minutes.
  7. Concentrate sample for 1-2 hours at 30° C in speed vac.

Hybridization

  1. Resuspend sample in 25-40 mL hybridization buffer (Tube Q).
  2. Place in 42° C water bath for 5-10 minutes.
  3. Heat probe mixture at 90° C for 2 minutes.
  4. At room temperature, add probe mixture to array. Cover with coverslip. Avoid air bubbles.
  5. Hybridize overnight at 60° C in water bath in hybridization with 2XSSC added to keep chamber moist.

Washes

These are all done at room temperature in a 50 mL conical tube.

Wash 1

  • 750 µL 20X SSC
  • 30 µL 10% SDS
  • Water to 30 mL

Wash just long enough for the coverslip to come off the slide.

Wash 2

  • 750 µL 20X SSC
  • 30 µL 10% SDS
  • Water to 30 mL

Wash for 15 minutes.

Wash 3

  • 90 µL 20X SSC
  • 30 µL 10% SDS
  • Water to 30 mL

Wash for 15 minutes.

Wash 4

  • 90 µL 20X SSC
  • Water to 30 mL

Wash for 15 minutes.
Spin dry slide and scan.


© 2012 The University of Texas MD Anderson Cancer Center