Hands-free Tissue Disruption— No Homogenization Needed
A Better Way to Disrupt Tissue
A Better Way to Isolate High Quality RNA
Figure 1. The MELT™ Procedure.
The integrity of RNA obtained using the MELT system is comparable to that obtained using two leading manufacturers' isolation kits (Figure 2A): single reagent, phenol-based lysis solution (Kit 1), followed by a glass fiber filter treatment, and a guanidinium isothiocyanate (GITC), glass filter-based method (Kit 2). However, RNA yields from the MELT protocols were up to three times greater (Figure 2B), and are generally higher due to the closed tube disruption step which limits handling and loss.
Figure 2. High Quality RNA with MELT™ Total RNA Isolation System. Total RNA was isolated from fresh mouse liver, kidney and brain tissue (~7 mg processed/reaction) using the MELT Total RNA Isolation System (Ambion), or one of two competitor kits, Kit 1 (single-reagent, phenol-based method, with an added glass filter cleanup step) or Kit 2 (GITC, glass fiber filter-based). RNA was isolated following the protocol for each method with the addition of a glass fiber filter purification step for Kit 1. (A) Agilent® 2100 bioanalyzer scans demonstrate the high quality total RNA isolated using the MELT Total RNA Isolation System. RIN = RNA Integrity Number, scaled from 1–10. (B) The averaged yield from 4 replicates with an input of ~7 mg of frozen or fresh mouse tissue.
High Quality RNA=Superior Results
Figure 3. qRT-PCR Analysis of Total RNA Isolated with MELT™ vs Competitor Total RNA Isolation Systems. Total RNA was isolated from fresh mouse liver and brain tissue (~7 mg tissue/reaction) using MELT Total RNA Isolation System (Ambion), or one of two competitor kits, Kit 1 (single-reagent, phenol-based method, followed by glass fiber filter isolation) or Kit 2 (GITC, glass fiber filter-based). The expression of 6 mouse genes, 3 of which are shown above, were quantitated using TaqMan® Gene Expression Assays (ABI). The data reveal < 1 Ct deviation between isolation methods for triplicate samples verifying that RNA isolated from the MELT System is comparable to that obtained from competitor kits. Assays were performed on a 7900 HT Sequence Detection System (ABI, standard cycling conditions) with MessageSensor™ RT Kit (Ambion).
Figure 4. Microarray Analysis of RNA from MELT™ Total RNA Isolation System. Total RNA was isolated from mouse liver, kidney, and brain tissue (~7 mg tissue/reaction) using the MELT Total RNA Isolation System (Ambion), or Kit 1 (single reagent, phenol-based method, followed by glass fiber filter isolation--the method currently recommended in Affymetrix's GeneChip® Expression Analysis Technical Manual), following the kit protocol. RNA (1 µg) was amplified with the MessageAmp™ II aRNA Amplification Kit (Ambion). aRNA was fragmented and hybridized to Mouse Genome 430A 2.0 arrays, scanned with GeneChip Scanner 3000, and data captured and analyzed on GeneChip Operating Software (Affymetrix). (A) Scatter Plot showing the high correlation between the MELT and Kit 1 methods with fresh mouse liver. A signal correlation plot showed 0.99 correlation between microarray data from samples prepared using each isolation method. (B) Scatter Plot demonstrating high correlation within the MELT method with RNA isolated from fresh mouse brain. (C) RNA was isolated from duplicate fresh mouse liver and kidney tissues as well as from fresh and frozen mouse brain tissue, using the two isolation methods as above. The data show Percent Present Calls for MELT vs Kit 1.
MELT Total RNA Isolation System
Heidi Peltier, Gary Latham • Ambion, Inc.