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

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

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

Labeling with Genisphere Kit

Mary Lee S. Ledbetter

College of the Holy Cross

Page 2: Labeling with Genisphere Kit Mary Lee S. Ledbetter College of the Holy Cross

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.

Page 3: Labeling with Genisphere Kit Mary Lee S. Ledbetter College of the Holy Cross

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)

Page 4: Labeling with Genisphere Kit Mary Lee S. Ledbetter College of the Holy Cross

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.

Page 5: Labeling with Genisphere Kit Mary Lee S. Ledbetter College of the Holy Cross

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+

Page 6: Labeling with Genisphere Kit Mary Lee S. Ledbetter College of the Holy Cross

                                                                                                                                                                                                                                                                                                                                

Sample data generated using this method

Long exposure

Short exposure

Page 7: Labeling with Genisphere Kit Mary Lee S. Ledbetter College of the Holy Cross

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