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).
Tissues and Cultured Cells Compatible with the MELT™ System.
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 . 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 .
Conveniently Store Lysates for Future Use
A major threat to RNA stability in tissues is RNase activity . 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 . 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.
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.
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.
Peltier H, Latham G (2005) Watch your tissue MELT into high quality total RNA. Ambion TechNotes 12(3):27.
Latham G, Peltier H (2005) MELT™ Total Nucleic Acid Isolation System: a new technology for hands-free tissue disruption, RNA preservation and total nucleic acid purification. Nature Methods 2(9):i–iii.
Krosting J, Latham G (2005) RNase activity in mouse tissues: classification, hierarchy, and methods for control. Ambion TechNotes 12(3):28.