Magnetism is the underlying principle of the automated nucleic acid isolation performed by the MagNA Pure system (Roche Diagnostics, Mannheim, Germany). The instrument has a completely closed housing as well as an automatic clot and tip loss detection. No filtration, centrifugation, or vacuum pumps are necessary, which minimizes the risk of cross-contamination. Up to 32 nucleic acid isolations (the eight-nozzle pipette head allows a variable number of samples from 1 to 32 per run) can be performed within one run. Extraction of human, viral, bacterial, and fungal DNA, RNA, and mRNA is possible. The protocol is applicable to a broad variety of samples such as blood, cultured cells, biopsies, sputum, urine, stool, plant tissue, and food products. After the extraction procedure, isolated nucleic acids and PCR mixes can be pipetted automatically into PCR tubes, 96-well plates, LightCycler capillaries (Roche Diagnostics), or COBAS A-Rings (Roche Diagnostics). Sample information can be entered manually or via a barcode reader. Samples are loaded into nuclease-free sample cartridges with a capacity of 4x 8 wells. The initial sample volume is 20-1000 pL (or up to 5 x 106 cells or 10 mg of homogenized tissue); the dispensable volume is 5-1000 pL with a 2-3% variance.
For the isolation of DNA or total nucleic acids, the sample material is initially incubated with a lysis buffer and proteinase K, and then magnetic glass particles are added. Nucleic acids bind to the surface of the magnetic glass beads and are thus separated from the sample remnants. For the extraction of mRNA, DNA is removed by DNase digestion. Streptavidin-coated magnetic beads and biotin-labeled oligo (dT) nucleotides are added to the lysed sample. The mRNA binds specifically to the glass beads through the oligo (dT) nucleotides.
In addition, since 2003, a smaller version named MagNA Pure Compact System is available. This system allows the extraction of nucleic acids from eight samples in parallel using bar-coded prefilled reagent cartridges and assembled tip trays.
Comparison of the MagNA Pure and a manual extraction protocol (phenol/chloroform) showed equivalent detection sensitivities for the extraction of Borrelia burgdorferi DNA from a variety of specimens such as urine, blood, and cerebral spinal fluid. All 80 positive samples (as determined by an independent method) were also tested positive by MagNA Pure extraction.1-8-1
Wolk et al. describe a protocol for the extraction of DNA from Encephalitozoon species. These species cause microsporidiosis, a disease of which the prevalence is likely to be underestimated because of the labor-intensive, insensitive, and nonspecific conventional methods used for the diagnosis of the spores in feces. The detection limit of the assay was 100-10,000 x lower compared to microscopy with trichrome stain.
Our group demonstrated that the MagNA Pure technique provides rapid automated DNA isolation from numerous pathogenic fungi revealing high sensitivity and purity. Although the fungal cell wall is highly resistant to mechanical, chemical, and enzymatical treatment, we could achieve a sensitivity of 1 colony forming unit/mL blood for Candida albicans.
In addition, there is a need for simple and sensitive assays for mRNA detection, e.g., to assess innate and adaptive immune responses or to determine different subpopulations of cells. The MagNA Pure protocol allowed extraction of mRNA from 32 samples in parallel within 1.5 hr, compared to at least 3 hr using manual methods. The amount (3-6 mg RNA from 106 PBMC) and purity (260:280 nm ratio in spectral photometer 1.8-1.9) of RNA were comparable or higher than those achieved by manual extraction.1-12-1
Finally, Williams et al. compared the BioRobot 9604 workstation to the MagNA Pure instrument, extracting _ ^ genomic DNA from 106 blood samples followed by genotyping for factor V Leiden. The comparison showed that both methods were similar in DNA yields (8.1 ±2.3 mg by MagNA Pure, 7.1 ±2.3 mg by BioRobot), although the DNA purity was higher in samples extracted by MagNA Pure (ratio 1.9±0.11 vs. 1.6±0.24). The extraction failure rate was 0.7% by MagNA Pure and 2.0% by BioRobot. Furthermore, with both assays the analysis of DNA extracted from sodium citrate and heparin anticoagulated blood samples was feasible. Finally, five heterozygous samples were analyzed in two separate runs. Statistical analysis using an F-test indicated that the DTms for the MagNA Pure were more consistent (p <0.001) which may be due to differences in sample purity.
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