MELT™ Total Nucleic Acid Isolation System | Hands-free Tissue Disruption

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.

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).

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].

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.

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.

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.