Aminoallyl-Labeling Of CDNA For Microarray Hybridization Protocol

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Title	Aminoallyl-Labeling Of CDNA For Microarray Hybridization

Overview

Microarray technology has enabled scientists to study thousands of genes simultaneously, identifying targets for metabolic regulation under a variety of conditions. In the case of cDNA arrays, the array itself is hybridized with the two different types of RNA or cDNA of interest (i.e. control and experimental) and each must be labeled for identification with different fluorescent dyes for detection. One of the most common methods of labeling involves the use of cyanin dyes (specifically, Cy 3 and Cy 5). This protocol offers a method for aminoallyl-labeling of cDNA using Cy3 and Cy 5 for microarray hybridization.

Reagents/Solutions

50X dNTP mix
All dNTPs should have stock solutions of 100 mM prepared prior to making the mix
For a volume of 40 ul, use the following volumes for dNTPs:
dATP: 10 ul (25 mM)
dCTP: 10 ul (25 mM)
dGTP: 10 ul (25 mM)
dTTP: 6 ul (15 mM)
aa-dUTP: 4 ul (10 mM)
Labeling Mix
Use the following concentrations and volumes to make the labeling mix:
Component Concentration Volume
First Strand Buffer 5X 6 ul
aa-dNTP mix 50X 0.6 ul
DTT 0.1M 3 ul
Reverse Transcriptase 50 U/ul 2 ul
Water 3 ul
Cy 3and Cy 5 Dyes
Cy3 and Cy5 dyes are dissolved in 48 ul DMSO, split in 3 ul aliquots and kept dry in the dark at 4C (preferably under vacuum) in the presence of dessicants.
Carbonate Buffer
0.1 M Carbonate Buffer, pH 9 is made fresh by mixing one volume 0.1 M NaCarbonate solution (10.6 g/L) with 20 volumes of 0.1 M Bicarbonate (8.4 g/L).
Reagents:
Superscript II Reverse transcriptase (Invitrogen, Carlsbad, CA)
Cy 3 Monoreactive Dye Pack (Amersham Bioscience, Piscataway, NJ)
Cy 5 Monoreactive Dye Pack (Amersham Bioscience, Piscataway, NJ)
Oligo(dT)12-18 (Invitrogen, Carlsbad, CA)
10 mM Aminoallyl-dUTP (Fermentas, Hanover, MD)
100 mM dATP, 100 mM dCTP, 100 mM dGTP, 100 mM dTTP (Amersham Bioscience, Piscataway, NJ)
Microcon YM-30 filters (Millipore)
QIAquick PCR Purification Kit (Qiagen, Billerica, MA)

References

Randolph, J. B., and Waggoner, A. S. Stability, specificity and fluorescence brightness of multiply-labeled fluorescent DNA probes. (1997) Nucleic Acids Res 25(14), 2923-9.
Brumbaugh, J. A., Middendorf, L. R., Grone, D. L., and Ruth, J. L. Continuous, online DNA sequencing using oligodeoxynucleotide primers with multiple fluorophores. (1988) Proc Natl Acad Sci U S A 85(15), 5610-4.
Hughes, T. R. et al. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. (2001) Nat Biotechnol 19(4), 342-7.
Exploratory and Confirmatory Gene Expression Profiling of mac1delta De Freitas, J.M., Kim, J.H., Poynton, H., Su, T., Wintz, H, Fox, T., Holman, P., Loguinov, A. Keles, S. van der Laan, M. and Vulpe, C. J. (2004) Biol. Chem. Vol. 279:4450-4458

Tips

This method works well using total RNA but it can be used to label polyA+ RNAs, as well. Priming of the reverse transcription can also be done using random 15-mer oligonucleotides or an equal mix of both random 15-mer and oligo-dT. If the probe is hybridized to an oligonucleotide array it is better to use random primers or a mix of oligo-dT and random primers in Step 1. From Step 12, the tubes should not be exposed to direct light to prevent fading of the fluorescence.