Article

Amine-modified random primers to label probes for DNA microarrays

Laboratory of Genetics, National Institute of Mental Health, Bethesda, MD 20892, USA.
Nature Biotechnology (Impact Factor: 39.08). 08/2002; 20(7):738-42. DOI: 10.1038/nb0702-738
Source: PubMed

ABSTRACT DNA microarrays have been used to study the expression of thousands of genes at the same time in a variety of cells and tissues. The methods most commonly used to label probes for microarray studies require a minimum of 20 microg of total RNA or 2 microg of poly(A) RNA. This has made it difficult to study small and rare tissue samples. RNA amplification techniques and improved labeling methods have recently been described. These new procedures and reagents allow the use of less input RNA, but they are relatively time-consuming and expensive. Here we introduce a technique for preparing fluorescent probes that can be used to label as little as 1 microg of total RNA. The method is based on priming cDNA synthesis with random hexamer oligonucleotides, on the 5' ends of which are bases with free amino groups. These amine-modified primers are incorporated into the cDNA along with aminoallyl nucleotides, and fluorescent dyes are then chemically added to the free amines. The method is simple to execute, and amine-reactive dyes are considerably less expensive than dye-labeled bases or dendrimers.

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Available from: Michael Brownstein, Dec 28, 2014
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    • "The labelling system is another major factor affecting microarray hybridization efficiency (Grigorenko, 2002). Xiang et al. (2002) and Bystricka et al. (2005) considered that indirect labelling of cDNA was more effective and less expensive than direct labelling. However, in this study, in which used PCR products were used, both labelling systems provided almost identical positive/negative hybridization patterns, although indirect labelling provided generally higher fluorescent intensities and slightly higher sensitivity than direct labelling. "
  • Source
    • "The labelling system is another major factor affecting microarray hybridization efficiency (Grigorenko, 2002). Xiang et al. (2002) and Bystricka et al. (2005) considered that indirect labelling of cDNA was more effective and less expensive than direct labelling. However, in this study, in which used PCR products were used, both labelling systems provided almost identical positive/negative hybridization patterns, although indirect labelling provided generally higher fluorescent intensities and slightly higher sensitivity than direct labelling. "
  • Source
    • "The labelling system is another major factor affecting microarray hybridization efficiency (Grigorenko, 2002). Xiang et al. (2002) and Bystricka et al. (2005) considered that indirect labelling of cDNA was more effective and less expensive than direct labelling. However, in this study, in which used PCR products were used, both labelling systems provided almost identical positive/negative hybridization patterns, although indirect labelling provided generally higher fluorescent intensities and slightly higher sensitivity than direct labelling. "
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