Thorough tissue disruption is essential for high RNA quality and yield. RNA that is trapped in intact cells is removed with cellular debris and is unavailable for subsequent isolation. Disruption is usually achieved by mechanical methods or by grinding the tissue. Physical dissociation of tissue, however, is cumbersome, low-throughput, and requires cleaning of equipment between samples. Open tube mechanical homogenization can also present biohazards through aerosols released from the sample.
The novel Multi-Enzymatic Liquefaction of Tissue (MELT) Total Nucleic Acid System from Ambion is a simpler and safer alternative. This technology enables hands-free tissue lysis through the use of powerful catabolic enzymes and a potent small molecule RNase inhibitor. RNA integrity obtained using the MELT system was comparable to that obtained using two leading manufacturers' isolation kits: 1) single reagent, phenol-based lysis solution, followed by glass fiber filter (GFF) treatment, and 2) a guanidinium isothiocyanate (GITC), GFF-based method. (Figure 3a).
However, RNA yields from the MELT protocols were up to three times greater (Figure 3b), in part due to closed tube disruption, which limits losses from tissue handling.
Unlike conventional RNA purification reagents, MELT proteases irreversibly inactive RNases by digesting them to peptide fragments. Figure 4 shows that intact RNA can be purified from MELT lysates even after 10 days of storage at room temperature while RNA from a GITC-based lysate was highly degraded within three days of room temperature storage.