Reverse Transcription in the Presence of Dideoxynucleotides to Increase the Sensitivity of Expression Monitoring with cDNA Arrays

Abstract

Complex cDNA targets conforming to the nomenclature proposed by Duggan et al. (Nat. Genet. 21 [1 Suppl.]:10–14) (where the immobilized nucleic acid is called “probe” and free nucleic acid is called “target”) were prepared by priming reverse transcription of mRNA with oligo-(dT)-primers or random oligonucleotide primers in the presence or absence of dideoxynucleotides. These targets were then hybridized to high-density filters spotted with 1339 cDNA clone inserts from a skeletal muscle library. The addition of dideoxynucleotides was found to significantly improve the efficiency and reproducibility of the reverse transcription reaction, and consequently, to improve detection of the least abundant transcripts in expression profiling experiments.