MagNA Pure LC RNA Isolation Kit III (Tissue)
Kit for the isolation of total RNA from fresh-frozen mammalian tissue and formalin-fixed, paraffin-embedded (FFPE) sections using the MagNA Pure LC Instruments.
For general laboratory use.
- Isolate high-quality total RNA from fresh, fresh-frozen, and RNAlater-stabilized tissues, as well as formalin-fixed, paraffin-embedded (FFPE) tissue sections.
- Obtain undegraded pure total RNA suitable for all possible downstream applications or long-term storage at -60°C or below.
- Total RNA is efficiently DNase-treated to prevent signal degradation due to residual traces of genomic DNA
- Wash Buffer I, 2 x 100 ml
- Wash Buffer II, 3 x 100 ml
- Binding Buffer, 100 ml 4. DNase I, RNase-free, 6 glass vials with lyophilizate
- DNase Incubation Buffer, 70 ml 6. Magnetic Glass Particles (MGPs), 6 glass vials with 8 ml MGP Suspension each
- Proteinase K, 6 glass vials with lyophilizate
- Proteinase K Buffer, 50 ml
- Elution Buffer, 100 ml
- Tissue Lysis Buffer, 2 x 70 ml
- Paraffin Homogenization Enzyme, 1 vial with lyophilizate
- Paraffin Homogenization Buffer, 1 x 20 ml
For general laboratory use.
The MagNA Pure LC RNA Isolation Kit III (Tissue) is designed to isolate high-purity total RNA from flash-frozen or formalin-fixed, paraffin-embedded human and other animal tissue samples on the MagNA Pure LC Instrument. Unfrozen tissue, stabilized by specific reagents (e.g., RNAlater), can also be used. The RNA purified from fresh tissue can be used in RT-PCR on the LightCycler® Carousel-Based Systems, the LightCycler® 480 System, standard thermal block cyclers, or other typical downstream applications in gene expression analysis. The quality of RNA isolated from formalin-fixed, paraffin-embedded tissue sections is suitable for relative quantifications of mRNA by RT-PCR, especially on the LightCycler® Instruments. Purified RNA is free of any PCR inhibitors. The purified RNA is also an ideal starting material for array experiments.
Number of isolations/sample type:
- 192 isolations (6 × 32) from 1 to 10 mg flash/frozen or unfrozen, stabilized mammalian tissue
- 96 isolations (3 × 32) from 5 to 20 μm sections of formalin-fixed, paraffin-embedded (FFPE) mammalian tissue
For details, see - Instructions for Use
To perform RNA isolations with the MagNA Pure LC RNA Isolation Kit III (Tissue), new protocols for the MagNA Pure LC Software must be installed.
The names "RNA III Tissue Fresh-Frozen" or "RNA III Tissue Paraffin" should appear in the protocol selection area of the "Sample Ordering" screen of the MagNA Pure LC Software.
If not previously installed, order the protocol (Cat. No. 03 356 698 001 for Software 2.11 (or lower); Cat. No. 03 356 701 001 for Software 3.0) on floppy disc free of charge. For additional details, contact your local Roche representative.
The tissue samples or paraffin sections are first homogenized using the MagNA Lyser Instrument (or other suitable homogenization devices) together with the Tissue Lysis Buffer included in the kit. Homogenization of formalinfixed, paraffin-embedded tissue sections is performed by an overnight incubation with Proteinase K. Sample lysates are then transferred to the MagNA Pure LC Instrument. A Proteinase K step enhances the cell lysis and digests the proteins. The RNA then binds to the surface of the added Magnetic Glass Particles (MGPs) under chaotropic-salt conditions. Unbound substances are removed by several washing steps. The purified RNA is then eluted from the MGPs, ready for PCR.
The principle steps of a MagNA Pure LC total RNA isolation procedure are:
- The sample material is placed into the wells of the Sample Cartridge.
- Lysis/Binding Buffer is added to the sample, resulting in complete cell lysis and release of RNA. RNases are denatured.
- RNA binds to the silica surface of the added MGPs due to the chaotropic salt conditions, isopropanol, and the high ionic strength of the Lysis/Binding Buffer.
- Genomic DNA is removed by incubation with DNase I.
- RNA is rebound to the particles by addition of isopropanol. MGPs with bound RNA are then magnetically separated from the residual lysed sample.
- MGPs with bound RNA are washed repeatedly with Wash Buffer to remove unbound substances like proteins (nucleases), cell membranes, PCR inhibitors such as heparin or hemoglobin, and to reduce the chaotropic salt concentration.
- Again MGPs with bound RNA are magnetically separated from the Wash Buffer containing residual sample debris.
- The purified RNA is eluted at +70°C from the MGPs in the wells of the Elution Cartridge, whereas the MGPs are retained in the reaction tip and discarded.
RNA is isolated from frozen mouse liver and formalin-fixed paraffin sections, using the appropriate purification protocol. Quality of the purified RNA is checked using gel electrophoresis, OD260/280 and RT-PCR/PCR analysis on the LightCycler® Carousel-Based System.
The kit components are tested for the absence of RNases.