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No Homogenization Required
Watch Your Tissue MELT into
High Quality Total RNA
• Suitable for both RT-PCR
and microarray gene expression analyses
• Hands-free tissue disruption in minutes: no
grinding or polytron required
• Protect and preserve RNA
in lysates for more than one week at ambient temperature
A Better Way to Disrupt Tissue
Physical dissociation of tissue is cumbersome,
low throughput, and potentially hazardous when samples are disrupted
in open tubes. Ambion’s Multi-Enzymatic Liquefaction of
Tissue (MELT™) Total RNA Isolation System (patent pending)
is a simpler and safer alternative to traditional RNA isolation
procedures that also allows high throughput tissue processing.
The MELT System is a hands-free technology for the rapid digestion
of fresh or frozen tissue. No homogenization is required. Samples
can be lysed easily in a closed-tube format without cross-contamination.
Up to 10 mg of tissue can be digested at room temperature using
a novel formulation that includes a potent RNase inhibitor and
a cocktail of powerful catabolic enzymes. Following digestion,
the RNA is purified using a streamlined process based on Ambion’s
patent pending MagMAX magnetic bead procedure and a special
vortex adapter. RNase-free DNase is provided to degrade genomic
DNA, and the RNA can be eluted from the beads in as little as
20 µl.
RNA Preservation
A major threat to RNA stability in tissues is RNase activity
(see RNase Activity in Mouse Tissue: Classification, Hierarchy,
and Methods for Control). Unlike harsh chaotropes
such as guanidine, the MELT reagents digest tissue to the molecular
level, reducing problematic RNases to peptide fragments that
can no longer degrade RNA. Once digestion is complete, the RNA
is highly stable and can be stored at room temperature for more
than one week without a significant loss in RNA stability. As
shown in Figure 1, a comparison of the MELT System to a common
guanidine (GITC)-based lysis method reveals that intact RNA can
be purified from MELT lysates even after 10 days of storage at
room temperature (22–25°C). Unlike RNA isolated with the MELT
System, RNA in the guanidine-based lysate was highly degraded
within three days of room temperature storage.
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Figure
1. RNA Preservation with the MELT™ Total
RNA Isolation System. MELT-liver and GITC-liver
lysates were incubated at room temperature (22–25ºC)
over 10 days. A fraction of the lysate was removed, and
the RNA purified at the time points noted. Agilent® 2100
bioanalyzer gel images of 28S and 18S rRNA bands demonstrate
intact RNA isolated from MELT lysates stored at room
temperature for several days. MELT=5 mg of frozen mouse
liver digested with the MELT System. GITC=5 mg of frozen
mouse liver disrupted in guanidinium isothiocyanate-based
buffer. ND=Not Determined: Agilent bioanalyzer software
unable to define 28S and 18S rRNA peak areas for RIN
or ratio quantitation. RIN=RNA Integrity Number, which
is based on the entire Agilent electropherogram (range:
0=highly degraded to 10=highly intact).
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High Quality MELT RNA is Suitable for Many Applications
RNA isolated from fresh or flash-frozen tissues
with the MELT System is appropriate for both quantitative and
endpoint RT-PCR. Following total RNA isolation and purification,
cDNA products as long as 8 kb can be amplified
(Figure 2). This system is ideal for mouse and human studies, because <2%
of all known mouse and human transcripts are longer than 8 kb (Dr. Xiaowei
Wang, Ambion, personal communication). A comparison of the MELT System with
another manufacturer’s kit (Kit 1: guanidinium-based procedure) reveals
that amplicons of 0.5, 1.2, 5.0, and 8.0 kb length can be successfully synthesized
from RNA purified using the MELT System, which indicates that long, intact
transcripts are represented in MELT RNA. In contrast, the 5.0 and 8.0 kb products
were much less pronounced using RNA isolated with Kit 1. For additional data
and representative results from microarray experiments using MELT lysates,
see Hands-free
Tissue Disruption.
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Figure
2. Amplicons as Long as 8 kb
Effectively Synthesized from Total RNA Isolated with
MELT™ Total RNA Isolation System. Total
RNA was isolated from ~7 mg of freshly collected mouse
liver and brain tissues using 2 different methods:
guanidinium isothiocyanate + glass fiber filter-based
procedure (Kit 1-GITC) or the MELT Total RNA Isolation
System. Total RNA (1 µg) was reverse
transcribed using Ambion’s ArrayScript™ RT and oligo(dT) for 1 h at 42ºC. The
RT step was followed by 40 PCR cycles using a hotstart
Taq polymerase. All RT minus controls were negative
for the specific products. Actb=b-actin, Casp3=Caspase-3, PKcs=Protein Kinase C, Fasn=Fatty Acid Synthase.
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MELT Total RNA Isolation System
Each kit contains sufficient reagents to process 50 samples
(<10 mg each). 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.
Scientific Contributors
Heidi Peltier, Gary Latham • Ambion, Inc.
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