Watch Your Tissue MELT into High Quality Total RNA
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.
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).
High Quality MELT RNA is Suitable for Many Applications
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.
Heidi Peltier, Gary Latham • Ambion, Inc.