Oligotex mRNA Kits

For low-throughput mRNA purification from total RNA, and for in vitro transcript cleanup

Features

  • 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 mRNA Mini Kit (12)

Cat. No. / ID: 70022

For 12 mRNA minipreps: 200 µl Oligotex Suspension, Small Spin Columns, Collection Tubes (1.5 ml), RNase-Free Reagents and Buffers
Column type
Midi
Mini
Oligotex mRNA Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

Oligotex mRNA Kits enable bead-based mRNA purification from total RNA and cleanup of poly A+ in vitro transcripts. Each Oligotex mRNA Kit includes Oligotex resin (oligo-dT beads) for binding poly A+ mRNA, and spin columns for washing and eluting bound mRNA.

Performance

The Oligotex mRNA procedure enables isolation of mRNA from less than 0.25 mg up to 1 mg of total RNA starting material (see table “Purification of mRNA from total RNA using the Oligotex mRNA procedure”).

Poly A+ mRNA purified with Oligotex resin is suitable for RT-PCR (see figure " RT-PCR of beta-actin mRNA") and poly A+ mRNA isolated from total RNA with Oligotex resin has been used for Northern, dot, or slot blotting (see figure " Isolation of mRNA from total RNA").

Purification of mRNA from total RNA using the Oligotex mRNA Mini and Midi procedure

Amount of starting material
(total RNA) per prep 
Maximum number of preps
per Oligotex mRNA Mini Kit*
Maximum number of preps
per Oligotex mRNA Midi Kit*
≤0.25 mg 12 (13)† 12 (28)†
0.25—0.5 mg 6 12 (14)†
0.5—0.75 mg 4 12 (14)†
0.75—1 mg 3 12
See figures

Principle

Oligotex mRNA Kits contain spin columns and all necessary reagents and buffers for isolation of pure poly A+ mRNA. The Oligotex low-throughput mRNA purification procedure enables isolation of pure poly A+ mRNA from total RNA in as little as 30 minutes, with >90% recovery. Isolation of poly A+ mRNA from total RNA usually provides slightly greater enrichment than direct isolation from cells or tissues, since there is no risk of interference by other cellular components.

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 oligo-dT beads 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. mRNA recoveries are consistently greater than 90%. The Oligotex bead-based mRNA 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. Poly A+ mRNA is immediately ready for all standard applications, as well as for sensitive applications such as expression array, differential display, cDNA library construction, microinjection, and SAGE technology.

See figures

Procedure

Optimized Oligotex purification procedures are convenient and straightforward (see flowchart "Oligotex mRNA procedure"). Total RNA preparations 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, including:

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

Comparison of Oligotex mRNA Kits

Features Oligotex mRNA Mini Kit Oligotex mRNA Midi Kit
Applications PCR, real-time PCR, microarray, transfection PCR, real-time PCR, microarray, transfection
Elution volume 20–100 µl 20–100 µl
Format Spin column Spin column
Main sample type Total RNA Total RNA
Processing Manual (centrifugation) Manual (centrifugation)

Purification of total RNA, miRNA,
poly A+ mRNA, DNA or protein

Poly A+ mRNA Poly A+ mRNA
Sample amount <250 µg 250 µg – 1.0 mg
Stabilization Yes Yes
Technology Polystyrene-latex particles Polystyrene-latex particles
Time per run or per prep 33–50 minutes 32–50 minutes
Yield >90% recovery >90% recovery

Supporting data and figures

Resources

User-Developed Protocols (1)
The procedure has been used successfully by customers for regeneration of Oligotex resin from spin columns following elution of poly A+ mRNA.
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 

FAQ

How can I decrease ribosomal RNA (rRNA) contamination using Oligotex?

The standard Oligotex procedures provide significant enrichment of poly A+ RNA. However, for a somewhat higher enrichment of mRNA, the following enrichment steps can be performed:

  • For Oligotex mRNA protocols: following step 5, add equal amounts of RNase-free water and Buffer OBB. (If following the Oligotex mRNA Protocol using Oligotex Direct mRNA Buffers, use 1 part Buffer OL1 plus 4 parts Buffer ODB). Mix the contents thoroughly by pipetting or flicking the tube. Repeat steps 3–5, and then continue the relevant protocol with step 6.
  • For Oligotex Direct mRNA protocols: for applications where highly purified mRNA is necessary (e.g., cDNA library construction), the full protocol rather than the shortened protocol is recommended. To clean up a sample with too much rRNA, do another round of purification following the optional protocol in Appendix D of the Oligotex handbook 'Oligotex mRNA Protocol Using Oligotex Direct mRNA Buffers', or use one of the Oligotex mRNA Kits.

Additional general tips:

  1. Add 0.5% SDS or LDS to the buffers in hybridisation and wash steps
  2. Double the volume of binding buffers without changing the amount of Oligotex Suspension
  3. Carry out binding/hybridisation with 300-400 mM NaCl (instead of 500 mM)
  4. Wash with 100 mM NaCl (instead of 150 mM)
  5. Use 10 mM of EDTA in the binding buffer (instead of 1 mM)
FAQ ID -642
What is the composition of buffer OW2?

The composition of Buffer OW2 is:

  • 10 mM Tris-Cl, pH 7.5
  • 150 mM NaCl
  • 1 mM EDTA

Buffer OW2 is the wash buffer used in Oligotex Kits for the isolation of poly-A mRNA from total RNA, or directly from cultured cells or tissues.

FAQ ID -419
Is mRNA isolation necessary for sensitive RT-PCR?
Usually not. Since RT-PCR is extremely sensitive, as little as 10–200 ng total RNA is sufficient for each 25–50 µl reaction mix, depending on the RT system. For abundant mRNA species, it is possible to use even less than 10 ng total RNA. For rare mRNA species, use a sequence-specific primer in the RT reaction to increase sensitivity. RNA content in various cells and tissues can be found here.
FAQ ID -111
What is the composition of buffer OBB?

The composition of Buffer OBB is:

  • 20 mM Tris·Cl, pH 7.5
  • 1 M NaCl
  • 2 mM EDTA
  • 0.2% SDS

Buffer OBB is the binding buffer used in the Oligotex mRNA Kits.

FAQ ID -421
How can I purify mRNA from total RNA using an Oligotex Direct mRNA Kit?
The Oligotex Direct mRNA Kits can be used to isolate poly A+ mRNA from total RNA by following Appendix D Protocol "Oligotex mRNA Protocol Using Oligotex Direct mRNA Buffers" in the Oligotex Handbook.
FAQ ID -201
What is the composition of buffer OCL?

The composition of Buffer OCL is:

  • 10 mM Tris-Cl pH 7.5
  • 140 mM NaCl
  • 5 mM KCl 1% Nonidet P-40*

Add before use (optional): 1000 U/ml RNase inhibitor; 1 mM DTT

*Nonidet P-40 is no longer manufactured. It can be replaced with Nonidet P-40 Substitute (Fluka, cat. no.74385) or Igepal® CA-630 (SIGMA, cat. no. I 3021).

Buffer OCL is used for direct mRNA purification from the cytoplasm of cultured cells with the Oligotex Direct mRNA Kits.

FAQ ID -422
What has to be done to an RNA sample before loading it onto an Agilent Bioanalyzer?

For RNA isolated on the BioRobot EZ1 and BioRobot M48:

The RNA can be directly applied to the Agilent Bioanalyzer, since it is being denatured in the final protocol steps of these isolation procedures.

For RNA prepared with all other QIAGEN RNA Isolation Products:

We recommend to denature the samples in a water bath for 2 min at 70°C, and then place them directly on ice prior to loading them onto the Agilent Bioanalyzer.

 

FAQ ID -528
What is the composition of buffer OEB?

The composition of Buffer OEB is:

  • 5 mM Tris-Cl, pH 7.5

Buffer OEB is the elution buffer used in the Oligotex mRNA and Oligotex Direct mRNA Kits for isolation of poly-A mRNA.

FAQ ID -420