Hands-free Tissue Disruption— No Homogenization Needed
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A Better Way to Disrupt Tissue
A critical first step in RNA isolation is to disrupt the tissue architecture and release the RNA. This step is typically achieved by mechanical methods using either a polytron or by grinding the tissue with a mortar and pestle. Physical dissociation of tissue, however, is cumbersome; low-throughput requires cleaning of equipment between samples, and is potentially hazardous when samples are disrupted in an open tube. 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. Samples can be lysed quickly and easily in a closed-tube format with minimal cross-contamination.
A Better Way to Isolate High Quality RNA
The MELT System is a hands-free technology for the rapid digestion of fresh or frozen tissue (Figure 1). 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 MagMAX™ magnetic bead procedure and a special vortex adaptor. RNA-binding magnetic beads are added to the MELT tissue lysate. The beads are then captured on a magnet, and cell debris and other contaminants are washed away. The beads are released from the magnet and treated with RNase-free DNase I to degrade genomic DNA. The fragmented DNA is removed by a subsequent wash step and the purified RNA is eluted from the beads in as little as 20 µl.
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.
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
The goal of any RNA isolation procedure is to recover an RNA population that faithfully mirrors the biology of the sample at the time of procurement. Ambion scientists have extensively validated the MELT technology to ensure that the purified RNA is functionally equivalent to RNA isolated using other popular methods. RNA purified using MELT technology, or two competing kits was first evaluated by real-time RT-PCR. Six distinct mRNAs were quantified across five different mouse tissues that were either freshly collected or flash-frozen. No significant difference was observed between samples isolated with MELT technology, or with competitor Kits 1 and 2 (data for 3 of the targets are shown in Figure 3). In addition, normalized microarray signal intensities obtained from samples isolated using MELT revealed higher Percent Present Calls (Figure 4C) and an excellent correlation (Figure 4A and B) compared to samples generated using the method recommended in Affymetrix's GeneChip® Expression Analysis Technical Manual (Kit 1 with an added cleanup step).
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.
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
The MELT Total RNA Isolation System contains enough reagents to isolate RNA from 50 samples, and ensures stabilization and recovery of high quality RNA that is ideal for gene expression analysis. DNase I and Buffer are also provided. A vortex with adaptor is necessary for sample mixing—the adaptor can also be obtained from Ambion (see sidebar at right).
Scientific Contributors
Heidi Peltier, Gary Latham • Ambion, Inc.
Scientific Contributors
Heidi Peltier, Gary Latham • Ambion, Inc.
