Optimization of DNase I Removal of Contaminating DNA from RNA for Use in Quantitative RNA-PCR

Abstract

In competitive RNA-PCR studies, contaminating DNA can produce incorrect results because of its potential to act as a second competitor. Preliminary studies using published methods for DNase I digestion of DNA as a contaminant of RNA, followed by thermal inactivation of the enzyme at 95°C for 5 min before reverse transcription and PCR, suggested that the mRNA was also affected by these treatments. This investigation was undertaken to optimize DNase I treatment of RNA with respect to DNA removal and mRNA preservation. Competitive RNA-PCR of DT-diaphorase transcript was used to quantitate the effects of the various treatments. Other transcripts with varying initial concentrations were visually compared to ensure that the effects observed were not unique to specific mRNAs. With 1 U of DNase I/mg RNA, thermal denaturation of the enzyme at 75°C for 5 min preserved nearly all of the mRNA. Thermal denaturation at 95°C for 5 min inactivated approximately 80% of the mRNA, whereas heating at 55°C for 10 min did not completely denature the DNase I. For RNA-PCR of every transcript investigated, incubation of 1mg RNA with 1 U of DNase for 30 min at 37°C followed by heat-denaturation of the enzyme for 5 min at 75°C was sufficient to destroy all the contaminating DNA, while completely preserving the respective mRNAs. This treatment is highly recommended as a routine step in RNA-PCR and particularly with competitive RNA-PCR with human breast tissue samples (and presumably other human tissues), which are often contaminated with small amounts of genomic DNA.