Oligotex Direct mRNA Mini Kit

Oligotex Direct mRNA kits are available for isolation of mRNA from as few as 100 cells to as many as 1 x 10⁸ cells (see Table 1), or from less than 10 mg of tissue up to 1000 mg of tissue (see Table 2).

Poly A⁺ mRNA purified from tissues or cells is suitable for use in downstream applications such as RT-PCR (see figure "RT-PCR of beta-actin mRNA").

Oligotex Direct mRNA Kits allow isolation of pure poly A⁺ mRNA directly from cells or tissues in as little as 1 hour (30 minutes with optional shortened protocols). The Oligotex Direct mRNA purification procedure utilizes rigorous denaturing lysis conditions to generate an immediate RNase-free environment for the isolation of intact mRNA. Oligotex Direct mRNA Kits contain a guanidine-thiocyanate–based buffer for efficient cell and tissue lysis, protein denaturation, and RNase inactivation, ensuring isolation of intact mRNA. The optimal design of Oligotex particles, in combination with RNA-protecting lysis and hybridization conditions, results in efficient purification of poly A⁺ mRNA in a short time with greater than 90% recovery. Poly A⁺ mRNA purified with Oligotex technology does not require further purification and is immediately ready for any standard application.

Oligotex resin consists of polystyrene–latex particles of uniform size (1.1 µm diameter) and a perfect spherical shape (see figure "Oligotex particles and oligo-dT cellulose") with dC₁₀T₃₀ oligonucleotides covalently linked to the surface. The size, composition, and surface structure of Oligotex particles have been optimized for uniform dispersion and minimal centrifugation time. The particles form a stable suspension that provides a large surface area for rapid and efficient binding of polyadenylic acids. The high capacity and accuracy of hybridization provides superior purification of poly A⁺ mRNA through fast and efficient hybridization.The Oligotex purification procedure minimizes loss of poly A⁺RNA, eliminates the risk of degradation by RNases, and requires minimal hands-on time. This makes it ideal for simultaneous handling of multiple samples.

Optimized Oligotex purification procedures are convenient and straightforward (see flowchart "Oligotex mRNA procedure"). Homogenized cell or tissue lysates are incubated with Oligotex resin, and Oligotex:mRNA complexes are collected by a brief centrifugation step. Spin columns, supplied in the Oligotex Kits, provide the most convenient handling, and optional batch protocols are also provided for certain applications. After washing, the mRNA is eluted in a small volume of Tris buffer or water. Alcohol precipitation is not required.

Poly A⁺ mRNA purified with Oligotex resin is ready for use in any downstream application, including:

• RT-PCR
• cDNA synthesis
• cDNA library construction
• Subtractive hybridization
• In vitro translation