- Isolate high-quality total RNA for sensitive downstream RT-PCR.
- Experience handsfree reproducibility, difficult to achieve using manual methods.
- Obtain scalable RNA yield and quantitative RT-PCR results that are a reflection of the amount of starting sample material purified.
- Wash Buffer I, 4 x 100 ml
- Wash Buffer II, 4 x 100 ml
- Lysis/Binding Buffer, 2 x 100 ml
- Magnetic Glass Particles (MGPs), 6 vials with MGP suspension
- DNase, 6 glass vials with lyophilizate
- DNase Buffer, 100 ml
- Isopropanol, 50 ml
- Proteinase K, 6 glass vials with lyophilizate
- Proteinase K Buffer II, 100 ml
- Elution Buffer, 100 ml
For general laboratory use.
The MagNA Pure LC RNA Isolation Kit - High Performance is designed to isolate high-purity total RNA from mammalian whole blood, blood cells, or cultured cells using the MagNA Pure LC Instrument. The purified RNA 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.
Number of isolations/sample type:
192 isolations (6 × 32) from
- 20 to 200 μl mammalian whole blood, of
- 1 × 103 to 1 x 106 mammalian blood cells (white blood cells [WBCs] or peripheral blood mononuclear cells [PBMCs]) or cultured cells
For details, see - Instructions for Use
To perform RNA Isolations with the MagNA Pure LC RNA Isolation Kit - High Performance, Software Version 3.0, new protocols must be installed.
The names "RNA HP Blood, RNA HP Cells or RNA HP Blood_external-lysis" should appear in the protocol selection on the "Sample Ordering" screen of the MagNA Pure LC Software. If not previously installed, order the protocols on floppy disc free of charge (Cat. No. 03 553 256 001).
The samples are lysed by incubation with a special buffer that contains chaotropic salt. Proteinase K digestion destroys remaining proteins and nucleases. Magnetic Glass Particles (MGPs) are added and the RNA is bound to their surfaces. DNA is degraded by incubation with DNase. Unbound substances are removed by several washing steps, and then the purified RNA is eluted.
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 was isolated from mammalian blood and cultured cells using the appropriate purification protocol. The quality of the purified RNA was determined using either agarose gel electrophoresis, OD260/280 readings or RT-PCR/PCR on the LightCycler® Instruments.