Oligotex mRNA Purification System

Oligotex Resin is designed for isolation of poly A+ mRNA from total RNA preparations or directly from animal cells and tissues using spin-column or batch procedures.

Features and benefits

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 Kit Options
Sample source	Recommended Oligotex Kit
Total RNA, in vitro transcripts	Oligotex mRNA Mini, Midi, and Maxi Kits
Animal cells or tissues	Oligotex Direct mRNA Micro, Mini, and Midi/Maxi Kits
—	Oligotex Suspension, Oligotex accessories

Principle

Oligotex resin consists of polystyrene–latex particles of uniform size (1.1 µm diameter) and a perfect spherical shape (see figure) 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. mRNA recoveries are consistently greater than 90%. 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.

Oligotex	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.

Procedure

Optimized Oligotex purification procedures are convenient and straightforward (see flowchart). 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. Homogenized cell or tissue lysates (Oligotex Direct mRNA procedure) or total RNA preparations (Oligotex mRNA procedure) 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. Neither procedure requires alcohol precipitation.

Applications

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

RT-PCR (see figure "RT-PCR of Beta-Actin mRNA")
cDNA synthesis
cDNA library construction
Subtractive hybridization
In vitro translation
Differential display
SAGE technology
Expression-array and expression-chip analysis
RNase and S1 nuclease protection
Primer extension
Northern, dot, or slot blotting (see figure "Isolation of mRNA from Total RNA")
Microinjection

Oligotex resin can replace soluble oligo-dT primers in cDNA synthesis reactions to yield cDNAs immobilized on Oligotex particles. Oligotex:cDNA complexes represent a reusable cDNA pool that can be directly used in subtractive hybridization to enrich low-abundance cDNAs of differentially expressed genes (1,2,3). The subtracted cDNA pool can then be used for library screening or construction.

Oligotex has also been used successfully for cleanup of poly-A tailed oligonucleotides (4).

RT-PCR of Beta-Actin mRNA

Reverse transcription was oligo-dT primed and PCR 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 & 2), using the Oligotex Direct mRNA Micro Kit. M: 123 bp ladder.

Isolation of mRNA from Total RNA

Formaldehyde agarose gel and northern blot (GAPDH-probed) of poly A+ mRNA isolated from total RNA from various samples using Oligotex mRNA Kits.

Cited References

1. Hara, E., Kato, T., Nakada, S., Sekiya, S, and Oda, K. (1991) Subtractive cDNA cloning using oligo(dT)30-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells. Nucleic Acids Res. 19, 7097.

2. Hara, E. et al. (1993) DNA-DNA subtractive cDNA cloning using oligo(dT)30-latex and PCR: identification of cellular genes which are overexpressed in senescent human diploid fibroblasts. Anal. Biochem. 214, 58.

3. Kuribayashi-Ohta, K., Tamatsukuri, S., Hikata, M., Miyamoto, C., and Furuichi, Y. (1993) Application of oligo(dT)30-latex for rapid purification of poly(A)+ mRNA and for hybrid subtraction with the in situ reverse transcribed cDNA. Biochim. Biophys. Acta 1156, 204.

4. Adam, G.I.R., Cui, H., Miller, S.J., Flam, F., and Ohlsson, R. (1996) Allele-specific in situ hybridization (ASISH) analysis: a novel technique which resolves differential allelic usage of H19 within the same cell lineage during human placental development. Development 122, 839.