GenElute™ Direct mRNA Miniprep Kits
SIGMA/DMN10 - sufficient for 10 purifications
Synonym: GenElute™ Direct mRNA Miniprep Kit; GenElute™ mRNA Kit; Gen Elute
Product Type: Chemical
Northern blot comparison of mRNA prepared directly from cells and tissues with GenElute Direct mRNA (DMN) & competitor kits.
Duplicate mRNA samples were prepared from 5 x 106 HEK 293 cells or 25-35 mg mouse liver with Sigma’s GenElute™ Direct mRNA Miniprep Kit or with several commercially available direct mRNA miniprep kits. A portion of each mRNA preparation equal to the amount from 1 × 106 cells or 10 mg liver was evaluated by Northern blot hybridization with 32P-labeled RNA probes for p53 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. Hybridization was detected and quantitated by scanning the blots with a PerkinElmer Instant Imager. Hybridization signals from each lane on the Northern blot, expressed as percent of the maximum signal for that probe, are plotted in the accompanying graphs.
Duplicate mRNA samples were prepared from 5 x 106 HEK 293 cells or 25-35 mg mouse liver with Sigma’s GenElute™ Direct mRNA Miniprep Kit or with several commercially available direct mRNA miniprep kits. A portion of each mRNA preparation equal to the amount from 1 × 106 cells or 10 mg liver was evaluated by Northern blot hybridization with 32P-labeled RNA probes for p53 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. Hybridization was detected and quantitated by scanning the blots with a PerkinElmer Instant Imager. Hybridization signals from each lane on the Northern blot, expressed as percent of the maximum signal for that probe, are plotted in the accompanying graphs.
Comparison of the preparation time, to isolate mRNA from total RNA or direct from cells or tissues, using GenElute™ mRNA Miniprep Kit and other commercially available kits. Each kit was prepared according to the procedure supplied by the vendor.
| Quality Level | 200  |
| storage temp. | 15-25°C |
| technique(s) | RNA purification: suitable |
| usage | sufficient for 10 purifications |
| Application: | The GenElute Direct Kit mRNA kit provides convenient procedures for isolating polyadenylated mRNA from previously prepared total RNA or directly from mammalian cells and tissues. |
| Features and Benefits: | • mRNA captured on oligo(dT) polystyrene beads in 10 minutes, with no mixing or rocking (Fig. 1) • Poly (A)+ mRNA isolated from total RNA in 40 minutes (Fig. 2) or 60 minutes directly from cells and tissues (Fig. 3) • Oligo(dT) polystyrene beads require fewer wash steps |
| Features and Benefits: | • Poly (A)+ mRNA isolated from total RNA in 40 minutes or 60 minutes directly from cells and tissues • Oligo(dT) polystyrene beads require fewer wash steps • mRNA captured on oligo(dT) polystyrene beads in 10 minutes, with no mixing or rocking |
| General description: | Click here to view the kit protocol |
| General description: | Procedures such as cDNA synthesis, expression profiling and others require separation of mRNA from the vastly more abundant rRNA and tRNA. The GenElute mRNA kits provide convenient procedures for isolating polyadenylated mRNA from previously prepared total RNA or directly from mammalian cells and tissues. For direct mRNA preparation, cells or tissues are disrupted with SDS/proteinase K digestion to release RNA and eliminate RNases. The kit uses oligo (dT) covalently linked to 1 μm polystyrene beads to capture polyadenylated mRNA by hybridization. The polystyrene beads remain suspended during hybridization, eliminating the need for mixing or rocking, as is common for cellulose or magnetic particles. Polystyrene was also chosen because oligo(dT) polystyrene beads yield cleaner mRNA with fewer stringent washing steps than does the more commonly used oligo(dT) cellulose (2 or 3 wash steps versus 10 or more). With the GenElute kits, mRNA-bead complexes are washed on a microcentrifuge spin filter, and eluted into 10 mM Tris-HCL, pH 7.5. Up to 107 mammalian cells or 40 mg tissue are lysed and homogenized, either with the filtration columns provided or with a mechanical homogenizer. RNase is eliminated during a 10 minute proteinase K digestion. Sodium chloride is added, and polyadenylated RNA is captured on oligo(dT) polystyrene beads during a 10 minute incubation. For further enrichment, RNA may be released from the beads into fresh lysis solution and recaptured with the original beads. After 3 washes in a spin column, purified mRNA is eluted in 100 μL of 10 mM Tris-HCl, pH 7.4. The purified mRNA is ready for Northern analysis, reverse transcription and PCR, labeling for arrays, and other common applications. |
| General description: | The GenElute™ Direct mRNA Miniprep kit provides a convenient format to isolate polyadenylated mRNA directly from mammalian cells and tissues. The direct mRNA isolation procedure is based on that of Badley. Up to 107 mammalian cells or 40 mg tissue are lysed and homogenized, either with the filtration columns provided or with a mechanical homogenizer. RNase is eliminated during a 10 minute proteinase K digestion. Sodium chloride is added, and polyadenylated RNA is captured on oligo(dT) polystyrene beads during a 10 minute incubation. For further enrichment, RNA may be released from the beads into fresh lysis solution and recaptured with the original beads. After 3 washes in a spin column, purified mRNA is eluted in 100 μl of 10 mM Tris-HCl, pH 7.4. |
| Legal Information: | GenElute is a trademark of Sigma-Aldrich Co. LLC |
| Other Notes: | For additional information, please see www.sigma-aldrich.com/mrn. |
| Other Notes: | Use for isolating mRNA directly from mammalian cells or tissues. |
| Symbol |   GHS05,GHS08 |
| Signal word | Danger |
| Hazard statements | H290 - H315 - H319 - H334 |
| Precautionary statements | P234 - P261 - P264 - P280 - P302 + P352 - P305 + P351 + P338 |
| Hazard Codes | Xn |
| Risk Statements | 36/37/38-42 |
| Safety Statements | 23-26-45 |
| RIDADR | NONH for all modes of transport |
| Flash Point(F) | Not applicable |
| Flash Point(C) | Not applicable |
| Storage Temp. | 15-25°C |
| UNSPSC | 41105501 |
| Components | Elution solution 1.5 mL; Filtration columns with tubes 10 ea; Lysis solution 20 mL; 5 M NaCl 1.5 mL; Oligo(dT)-polystyrene beads .3 mL; Proteinase K 5 mg; 40% Glycerol solution .6 mL; Spin columns with tubes 10 ea; Collection tube 10 ea; Wash Solution; Low Salt Wash Solution; Proteinase K from Tritirachium album, lyophilized powder, Molecular Biology, BioUltra, ≥30 units/mg protein; Sodium chloride solution, 5 M in H2O, BioReagent, Molecular Biology, suitable for cell culture |