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MELT™ Total Nucleic
Acid Isolation System
Hands-free Tissue Disruption
• Hands-free tissue disruption
in minutes: no grinding or polytron required
• Suitable for RT-PCR, PCR, and microarray gene expression analysis
• Isolate both total RNA and genomic DNA
• Protect and preserve RNA for days in tissue lysates at ambient temperature
A Better Way to Disrupt Tissue and
Isolate RNA and DNA
Ambion’s Multi-Enzymatic Liquefaction
of Tissue (MELT™) Total Nucleic Acid Isolation System (patent
pending) is a simpler and safer alternative to traditional RNA
and genomic DNA isolation procedures.
The MELT System permits the hands-free disruption of several
solid tissues in parallel without the need of tissue grinding
or a polytron, which can create toxic aerosols. Samples can be
lysed easily in a closed-tube format without cross-contamination.
Up to 10 mg of fresh or frozen tissue can be liquefied at room
temperature in 5–15 minutes using a combination of powerful catabolic
enzymes and a potent small-molecule RNase inhibitor. Following
digestion, the RNA or DNA is purified using a streamlined process
based on Ambion’s MagMAX™ magnetic bead technology (patent
pending). The purified RNA or DNA can be eluted from the beads
in a concentrated form in as little as 15–20 µl.
A Variety of Tissues can be Processed
in the MELT System
The MELT System is compatible with a range
of freshly procured or flash-frozen tissues. There is no lower
limit of tissue mass for processing, and tissues up to 10 mg
in size are suitable for digestion. More than a dozen commonly
studied human and rodent tissues, and cultured cells have been
used successfully with the MELT system (Figure 1).
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Figure
1. Tissues and Cultured Cells
Compatible with the MELT™ System.
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High Quality MELT RNA is Suitable
for Many Applications
The goal of any RNA isolation procedure is
to recover an RNA population that is highly representative of
the biological sample at the time of procurement. Ambion extensively
validated the MELT technology to ensure that the purified RNA
was functionally equivalent to traditionally isolated RNA. The
resulting RNA was tested by endpoint RT-PCR to amplify products
as long as 8 kb and real-time RT-PCR to quantify the expression
level of nearly a dozen different sentinel transcripts [1]. In
addition, microarray analyses that have interrogated >14,000
genes revealed the MELT system to be highly correlated with the
RNA isolation method recommended in the Affymetrix® GeneChip® Expression
Analysis Technical Manual [2].
Conveniently Store Lysates for Future
Use
A major threat to RNA stability in tissues
is RNase activity [3]. Unlike harsh chaotropes such as guanidine,
the MELT reagents digest tissue to the molecular level, preventing
reactivation of problematic RNases. Once the tissue digestion
is complete, the MELT lysate can be stored frozen at -20°C, -80°C,
or even at room temperature (for up to a week for many tissues)
without a significant loss in RNA stability [1]. Lysates can
be stored similarly for DNA isolation.
Isolate Both RNA and DNA
The MELT system is now available with the option
to isolate genomic DNA from solid tissues, including mouse tails.
Although conventional mouse tail DNA isolation procedures require
lengthy, overnight digestions, MELT provides both tissue digestion
and DNA isolation protocols that can be completed in just hours,
enabling same day results. DNA can be purified from MELT lysates
(Figure 2) with reagents provided in the current kit format,
along with RNase A (not included) for an optional RNase treatment
step. The purified DNA is suitable for both endpoint and real-time
PCR.
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Figure
2. DNA Isolation Using the
MELT™ Total Nucleic Acid Isolation System. (A) 1%
Agarose gel, ethidium bromide stained, displays DNA
isolated from various tissue types with the MELT System
(~500 ng of each sample per lane). (B) Real-time
PCR targeting GAPDH and FAS genomic sequences using
100 ng input RNA and TaqMan® technology (ABI).
Error bars indicate the range from duplicate reactions.
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MELT Total Nucleic Acid Isolation
System
Each kit contains sufficient reagents to process
50 tissue samples (<10 mg each) for either RNA or genomic DNA
isolation. A specially designed vortex adapter and either 6 tube
or 96 well magnetic stands are available separately for multi-sample
processing. The ability of the MELT system to achieve both tissue
digestion and RNA preservation enables samples to be safely processed,
conveniently stored, and readily transported at ambient temperatures
for a host of downstream analyses.
[Additional information]
Scientific Contributors
Heidi Peltier, Gary Latham • Ambion, Inc.
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