Labeling with Genisphere Kit

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Labeling with Genisphere Kit

• Mary Lee S. Ledbetter
• College of the Holy Cross

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• Advantages of direct labeling
• Involves fewer steps, and hence opportunities
  for variability to creep in
• Allows monitoring of the level of dye
  incorporation into the target molecules.
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• Disadvantages of direct labeling
• Requires a large amount of starting material (50
  µg per treatment)
• Introduces bulky fluorescent groups into the
  cDNA, which might interfere with synthesis or
  hybridization
• Suffers from bias toward one or the other dye.
• Requires that all materials be protected from
  light.

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Indirect labeling

• Uses the relatively small amino allyl group to
  make one of the nucleotides reactive, avoiding
  bias in synthesis and increasing yield.
• Addition of the dye by a coupling reaction after
  the cDNA is synthesized helps to avoid dye
  asymmetry favoring one of the two dyes during
  incorporation.
• However,
• Still need 50 µg of starting RNA
• Must take precautions to protect from light once
  dye is added (including during steps to clean up
  the reaction mix)

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3DNA dendrimer probes

• Procedure requires only 5 µg total RNA
• Fluorescence is added in the next-to-last step,
  making previous handling of samples much easier
• The signal is amplified tremendously through the
  structure of the dendrimers, so arrays tend to
  have low background and bright signals
• BUT
• Two hybridization steps are required, not one.
• Dyes may be subject to differential oxidation
  prior to scanning.

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One way to label cDNA (3DNA kit from Genisphere)
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Sample data generated using this method
                                                  
                                                  
                                                  
          
                                                  
                                                  
                                                  
          
Long exposure
Short exposure
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Two grids within a larger microarray showing
spots of various intensities and hues