Mouse liver total RNA was isolated according to protocol. RT-PCR reactions were performed using Ambion's RETROscript?Kit and 0.5 µg RNA. PCR reactions were performed with 5 µg RNA. 10 µl of each reaction was electrophoresed on a 2% agarose gel and stained with EtBr.
DNase I Treatment
In a recent informal survey of RT-PCR users, we found that the field is evenly divided by those users who believe that DNase I treatment solves the problem of genomic DNA contamination and those who feel that DNase I treatment is an unacceptable solution. Detractors claim that DNase I treatment and the subsequent inactivation steps compromise the performance of their RT-PCR reactions to an unacceptable degree. Much of the problem these users experience may be traced to the extreme temperatures used to inactivate the DNase I prior to reverse transcription. Huang, et al. (Biotechniques, (1996) 20:(6)1012-1020) report complete inactivation of DNase I by heat denaturation at 75°C for 5 minutes. Lower inactivation temperatures do not completely inactivate DNase I, while higher temperatures appear to damage the RNA template. DNase I treatment followed by heat inactivation is a simple enough technique for routine use in systems in which genomic DNA contamination is a problem. The use of high quality, RNase-free DNase is crucial. Two additional conventional methods of reducing contaminating genomic DNA from total RNA preparations are acid phenol extraction, which partitions DNA into the organic phase, and LiCl precipitation, which selectively precipitates RNA from solution (protein and DNA remain in the supernatant). A description of these techniques can be found in Ambion's Technical Bulletins #158 and #160. These techniques can be used after DNase I treatment to inactivate the enzyme and precipitate the RNA prior to reverse transcription. Finally, it should be noted that DNase I treatment neither relieves the investigator of the burden of sensible primer design, nor of the necessity to perform the appropriate "no-RT" controls.
In addition to the above techniques, researchers now have a new and convenient option for removal of DNA and DNase I from RNA samples. DNA-free™ DNase Treatment and Removal Reagents are designed for the removal of contaminating DNA from RNA samples and for the removal of DNase after treatment. As described above, DNase is typically inactivated by heat treatment, and can also be removed from treated preps by phenol extraction. Heat inactivation can present problems, however, as the temperature at which DNase is inactivated also catalyzes RNase-independent RNA strand scission in the presence of divalent cations. Phenol extraction is also avoided by researchers who do not want to work with phenol, or who worry about sample loss.
DNA-free avoids both methods of DNase I inactivation by supplying a novel DNase Removal reagent that effectively removes DNase and divalent cations from the reaction mixture. The DNase/cation removal step takes a mere three-minute incubation. No organic extraction, EDTA addition or heat inactivation is required.
The DNA-free DNase Treatment and Removal Reagents are provided with RNase-free DNase I, an optimized 10X Reaction Buffer, and the DNase Removal Reagent. The DNA-free Reagents are now also part of the RNAqueous™-4PCR Kit, combining the features and benefits of RNAqueous™ with those of DNA-free.
| Figure 4. RT-PCR Experiments Using Total RNA DNase-Treated Using DNA-free Reagents. Five µl of RNA samples isolated using Ambion's RNAqueous™ Kit were used as templates for reverse transcription reactions; 10% of the resulting cDNA was amplified by PCR using S15 primers. The lanes to the left of the markers are PCR reactions done without reverse transcription, demostrating the lack of genomic DNA contamination in these RNA samples. The lanes to the right of the markers show the S15 RT-PCR product from the indicated samples. |
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