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

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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 ...

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