Amine-modified random primers to label probes for DNA microarrays

Charlie C. Xiang, Olga A. Kozhich, Mei Chen, Jason M. Inman, Quang N. Phan, Yidong Chen, Michael J. Brownstein

Research output: Contribution to journalArticle

64 Scopus citations

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 μg of total RNA or 2 μg 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 μg 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.

Original languageEnglish (US)
Pages (from-to)738-742
Number of pages5
JournalNature Biotechnology
Volume20
Issue number7
DOIs
StatePublished - Jan 1 2002
Externally publishedYes

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ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Molecular Medicine
  • Biomedical Engineering

Cite this

Xiang, C. C., Kozhich, O. A., Chen, M., Inman, J. M., Phan, Q. N., Chen, Y., & Brownstein, M. J. (2002). Amine-modified random primers to label probes for DNA microarrays. Nature Biotechnology, 20(7), 738-742. https://doi.org/10.1038/nb0702-738