DescriptionThe polyA Spin™ Kit is a rapid and convenient alternative to traditional column chromatography for the isolation of full-length poly(A)+ eukaryotic messenger (mRNA) from samples of "total" RNA. Poly(A)+ RNA selection is made by affinity chromatography using spin columns prepackaged with NEB's Oligo (dT)25-Cellulose beads (NEB #S1408S). Prepared essentially by the method of Gilham (1), this solid support has found widespread use in the "batch" preparation of low abundance mRNA (2). Its high binding capacity and rapid hybridization kinetics make it an ideal support for chromatographic poly(A)+ RNA selection. Intact mRNA can be isolated in as little as forty minutes from multiple cell lysates or samples of "total" RNA by spin chromatography in a microcentrifuge.
Reagents sufficient for the isolation and subsequent precipitation of poly(A)+ RNA from eight samples of as much as 1.0 mg of "total" RNA are provided. The isolated RNA can be used for in vitro translation experiments, the preparation of cDNA libraries, Northern analysis, subtractive hybridization or differential display.
- Shorter Purification Time
- Reduced Isolation Volumes
- Maintains Resolution: When compared to a 100 mg column of high quality oligo (dT) cellulose in 1.0 mg total RNA sample range.
- microcentrifuge tubes containing oligo (dT)25 -cellulose beads
- Glycogen Solution
- Elution Buffer (S1560, S1550)
- Wash Buffer (15 ml)
- Low Salt Buffer
- 5 M NaCl
- 3M NaAC
- microcentrifuge spin columns
- sterile microcentrifuge tubes
- Elution Buffer
- Wash Buffer I
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
|microcentrifuge tubes containing oligo (dT)25 -cellulose beads|
|Glycogen Solution||10 mg/ml|
|Elution Buffer (S1560)||1X|
|Wash Buffer (15 ml)||1X|
|Low Salt Buffer||1X|
|5 M NaCl||500 mM|
|3M NaAC||300 mM|
|microcentrifuge spin columns|
|sterile microcentrifuge tubes|
|Wash Buffer I||1X|
Properties and Usage
Quality Assurance Statement
- Each lot of the polyA Spin mRNA Isolation Kit is assayed for its ability to isolate mRNA from rat liver total RNA.
- The polyA Spin mRNA Isolation Kit complements and serves as an alternative to traditional column chromatography isolation of mRNA. Each polyA Spin spin column has the same column performance as a standard 100 mg column of high quality oligo (dT)-cellulose in the 1.0 mg total RNA sample range. Thus the shorter purification times and reduced working volumes associated with spin chromatography are realized while maintaining column chromatography levels of performance. This is of particular importance in the isolation of low abundance messenger RNA (1). For total RNA samples greater than 1.0 mg initial "first round" isolation of poly(A)+ material should be done by column or "batch" (NEB #S1408S) chromatography (2). First round isolation of poly(A)+ RNA by either column or spin chromatography typically yields a product contaminated with poly(A)– RNA. PolyA Spin spin chromatography is ideal for "second round" purification, yielding material with an A260/280 of 1.9 or greater with minimal product loss. The protocol has been optimized for the isolation of poly(A)+ RNA from 0.1–1.0 mg of total RNA isolated from eukaryotic tissue or cells that contain between 1–5% mRNA. The amount of poly(A)+ RNA isolated will vary with the type of tissue or cells used.
- Gilham, P.T. (1964). J. Amer. Chem. Soc.. 86, 4982.
- Farrell, R.E. (1993). RNA Methodologies. 104-106.
- Jacobson, A. (1987). S. Berger and A. Kimmel(Ed.), Methods Enzymol.. 152, 254-257. San Diego: Academic Press.
The denaturing step is critical for adequate mRNA recovery so please, make sure your sample is thoroughly denatured before incubating it with the beads.
Centrifugation speed should never exceed 12,000 x g