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.
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.