Exploring Reverse Transcription for DNA MicroArrays

Abstract

We designed 96 un-modified 50-mer DNA oligonucleotides, one for every 2 kb of the longest human genes known: Titin, Nebulin and Obscurin, all expressed in human muscle (Gene length ~100kb). DNA oligonucleotide targets for positive (a-Actin) and negative controls (ß-Actin) for human muscle tissue were included and spotted on epoxide-coated glass slides at 1 - 50µM concentration to create a DNA microarray.

Oligo-dT, random and non-priming strategies were explored for reverse transcription (RT). Results were determined by microarray spot fluorescence analysis.

Our results suggest that random priming is the optimal method for expression analysis of long genes. This method of RT may be appropriate for alternatively spliced genes and for genes without unique probes in the 3’-region.