Amine-modified random primers to label probes for DNA microarrays

DNA microarrays have been used to study the expression of thousands of genes at the same time in a variety of cells and tissues(1-3). The methods most commonly used to label probes for microarray studies require a minimum of 20 mug of total RNA or 2 mug of poly(A) RNA(4,5). This has made it difficult to study small and rare tissue samples. RNA amplification techniques and improved labeling methods have recently been described(6-9). 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 mug 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.