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Oligotex Direct mRNA Midi/Maxi Kit

For mRNA purification from cells and tissues
  • High recovery of pure mRNA in as little as 30 minutes
  • No oligo-dT cellulose or ethanol precipitation
  • Flexibility for use with widely varying amounts of starting material
Oligotex Direct mRNA Kits enable purification of mRNA from cells and tissues. Each Oligotex Kit includes Oligotex resin for binding poly A+ mRNA, and spin columns for washing and eluting bound mRNA. For purification of mRNA from total RNA and cleanup of poly A+ in vitro transcripts, Oligotex mRNA Kits are available.
Cat No./ID: 72041
Oligotex Direct mRNA Midi/Maxi Kit (6/2)
€1,089.00
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For 6 mRNA midi or 2 maxipreps: 1 ml Oligotex Suspension, Large Spin Columns, Collection Tubes (1.5 ml), RNase-Free Reagents and Buffers
The Oligotex Direct mRNA Midi/Maxi Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Oligotex mRNA procedure.
1
RT-PCR of beta-actin mRNA.
Reverse transcription was oligo-dT primed and PCR was carried out using actin-specific primers. [A] Poly A+ mRNA was isolated from 0.25, 0.5, or 1 µg mouse liver total RNA (lanes 1–3), using the Oligotex mRNA Mini Kit. C: negative control. [B] mRNA was isolated from 103 or 104 HeLa cells (lanes 1 and 2), using the Oligotex Direct mRNA Micro Kit. M: 123 bp ladder.
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Oligotex particles and oligo-dT cellulose.
Scanning electron micrographs of Oligotex particles and oligo-dT cellulose at the same magnification. Note the uniform size and shape of Oligotex particles.
Performance

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").

Table 1. Specifications of Oligotex Direct mRNA for purification of mRNA from cells
Amount of starting material (number of cells) per prep Maximum number of preps per Oligotex Direct mRNA Mini Kit Maximum number of preps per Oligotex Direct mRNA Midi/Maxi Kit
100* –2 x 105 12 (14) 6 (14)
2 x 105–5 x 105 12 (14) 6 (14)
5 x 105–1 x 106 12 (14) 6 (14)
1 x 106–5 x 106 12 6 (14)
5 x 106–1 x 107 6 6 (14)
1 x 107–2 x 107 3 6 (9)
2 x 107–3 x 107 6
3 x 107–5 x 107 6
5 x 107–7 x 107 4
7 x 107–1 x 108 2
* Lowest amount tested.
Maximum number of preps per kit with purchase of additional spin columns. Additional 1.5 ml microcentrifuge tubes may be required.
Table 2. Specifications of Oligotex Direct mRNA for purification of mRNA from tissues
Amount of starting material (tissue) per prep Maximum number of preps per Oligotex Direct mRNA Mini Kit Maximum number of preps per Oligotex Direct mRNA Midi/Maxi Kit
≤10 mg 12 (14)* 6 (14)*
10–25 mg 12 (14)* 6 (14)*
25–50 mg 6 6 (14)*
50–100 mg 3 6 (9)*
100–150 mg 6
150–250 mg 6
250–500 mg 4
500–750 mg 2
750–1000 mg 2
* Maximum number of preps per kit with purchase of additional spin columns. Additional 1.5 ml microcentrifuge tubes may be required.
Principle

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 Asup>+ 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.

Procedure
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.
Applications

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

  • RT-PCR
  • cDNA synthesis
  • cDNA library construction
  • Subtractive hybridization
  • In vitro translation
Features
Specifications
Applications PCR, real-time PCR, microarray, transfection..
Elution volume 50–500 µl
Format Spin column
Main sample type Tissue, cells
Processing Manual (centrifugation)
Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein Poly A+ mRNA
Sample amount 1.0 g/1 x 10e8
Stabilization Yes
Technology Polystyrene-latex particles
Time per run or per prep 30–50 minutes
Yield >90% recovery

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Handbooks & Protocols (1)
For Purification of poly A+ RNA from total RNA, purification of poly A+ RNA directly, from cultured cells or tissues, and purification of polyadenylated in vitro transcripts 
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Kit Handbooks (1)
For Purification of poly A+ RNA from total RNA, purification of poly A+ RNA directly, from cultured cells or tissues, and purification of polyadenylated in vitro transcripts 
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User-Developed Protocols (2)
The procedure has been used successfully by customers for regeneration of Oligotex resin from spin columns following elution of poly A+ mRNA.
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The procedure has been successfully used for isolation of mRNA from adipose tissue by customers. Because of the nature of adipose tissue it cannot be used immediately in Oligotex Direct mRNA Protocols; simple centrifugation, however, eliminates the problem, and the procedure works fine.
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