|
TURBO DNA-free™ Kit
The Fastest,
Simplest, and Most Effective Way to Remove DNA Contamination
• Orders of magnitude better
DNA removal than with wild type DNase I
• 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 µg sample of spleen
RNA was then digested for 15 min at 37°C with 12 U of TURBO DNase
or 12 U of DNase I from Competitor P in a 100 µl reaction.
Reactions were then processed as instructed by the TURBO DNA-free
Kit Instruction Manual or by the competitor’s 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 µg of the RNA was then treated with either 12 U of TURBO DNase™ or DNase I from Competitor P in a 100 µl reaction. After DNase digestion, samples were processed by following the TURBO DNA-free or the competitor’s 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 competitor’s 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.
Scientific Contributors
Jon Kemppainen, Gary Latham • Ambion,
Inc.
back
to top
|
