Labeling with Genisphere Kit

1. Labeling with Genisphere Kit Mary Lee S. Ledbetter College of the Holy Cross

2. 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. • ??? • 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.

3. 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)

4. 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.

5. One way to label cDNA (3DNA kit from Genisphere): o x o o o x x o o o o o x o x o +

6. Sample data generated using this method Long exposure Short exposure

7. Two grids within a larger microarray showing spots of various intensities and hues