Perform DNA digestion and sample clean-up in just 20 minutes
Preserve RNA quality
DNase treatment is the method of choice for the removal of contaminating genomic DNA from RNA samples in preparation for RT-PCR. TURBO DNase (patent pending) is an engineered variant of DNase I with a 6-fold lower Km for DNA than wild type DNase I. As a result, TURBO DNase is a superior enzyme for removal of trace amounts of contaminating DNA from RNA preparations. Using the TURBO DNA-free Kit, DNA digestion and the subsequent removal of DNase and divalent ions from the RNA sample, can be accomplished within 20 minutes.
We compared the TURBO DNA-free Kit with a popular wild type DNase I enzyme (Competitor P). To highlight the benefits of TURBO DNase, total RNA was purified from mouse spleen, a notoriously DNA-rich tissue, using a procedure that results in significant contamination with genomic DNA. An 8 g sample of spleen RNA was then digested for 15 min at 37C with 12 U of TURBO DNase or 12 U of DNase I from Competitor P in a 100 l reaction. Reactions were then processed as instructed by the TURBO DNA-free Kit Instruction Manual o r by the competitors protocol. The treated RNA was subjected to real-time PCR to detect genomic DNA contamination that remained after DNase treatments. A GAPDH primer/probe set was used in triplicate reactions, and the extent of genomic DNA removal was calculated from the real-time PCR cycle threshold (Ct) values.
Elimination of DNA contamination by TURBO DNase, compared to no DNase treatment, shifted the real-time PCR results by >20 Ct values, representing >1.2 million fold reduction of DNA contamination. When compared to standard DNase I treatment, TURBO DNase decreased genomic DNA by an additional 22.6 fold (Figure 1). Indeed, one of the three TURBO DNA-free replicates was negative even after 40 cycles of PCR.
Figure 1. The TURBO DNA-free Kit Removes >20-fold more Genomic DNA than Competing Wild Type DNase I. RNA was isolated from mouse spleen using a procedure that results in significant contamination with genomic DNA. 8 g of the RNA was then treated with either 12 U of TURBO DNase or DNase I from Competitor P in a 100 l reaction. After DNase digestion, samples were processed by following the TURBO DNA-free or the competitors protocol. Triplicate samples of the treated RNA were subjected to real-time PCR using a GAPDH primer/probe set. Note that treatment with TURBO DNase shifted the average Ct threshold 4.5 cycles, equivalent to 22.6 fold reduction of genomic DNA contamination.
By comparison, the wild type DNase I removed 20 times less DNA; GAPDH was detected in all three replicates after 30 cycles. In addition, RNA LabChip analysis of the DNase-treated RNA revealed that TURBO DNA-free was far superior in preserving RNA quality (28S/18S rRNA=1.1) than the competitors method (28S/18S rRNA=0.36). The TURBO DNA-free Kit safeguards RNA quality using reagents that are more potent and much faster and simpler to use than competing products that include wild type DNase I.
Jon Kemppainen, Gary Latham Ambion, Inc.
Cat# Product Name Size 1907 TURBO DNA-free 50 rxns