Description
General description
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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.
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.
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
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
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
Other Notes
For additional information, please see www.sigma-aldrich.com/mrna.
Use for isolating mRNA directly from mammalian cells or tissues.
Legal Information
GenElute is a trademark of Sigma-Aldrich Co. LLC
