The size of eukaryotic mRNAs are ranging from 0.3 kb to over 30 kb. The abundance of mRNAs is from fewer than 5 copies to over
30,000 copies per eukaryotic cell. Polyadenylated mRNAs can be
separated from nonpolyadenylated transfer RNAs and ribosomal RNAs
that account over 95% of total cellular RNA. The general principle
is that an RNA-DNA hybrid is formed between the poly A+-tail and the oligo (dT)12-18-Cellulose at 400 mM Sodium chloride, and that excess rRNAs, tRNAs
and other small RNAs can be washed away at this ionic strength.
The major drawback of this procedure is the need to start with
total RNA on a milligram scale to avoid substantial losses during
chromatography, extraction with organics and precipitations.
MATERIALS AND SOLUTIONS
Regeneration and Preparation of Oligo(dT)-Cellulose
(1) Weigh 0.25 g oligo(dT)-cellulose in a 15 ml tube.
(2)Wash oligo(dT)-cellulose two times each with;
- 0.1 M NaOH ------------------------------- 10 ml
- DEPC-treated H2O ------------------------- 10 ml
- Elution buffer -------------------------------- 10 ml.
- Centrifuge for 5 minutes at 3,000 rpm each time to collect oligo(dT)-cellulose.
(3) Add 5 ml 2x Binding Buffer and 5 ml DEPC-treated H2O.
(4) Mix and equilibrate for 1-2 minutes.
For long term storage the matrix can be stored at 4oC in 1 X Binding Buffer + 0.02% Sodium azide (NaN3).
2 X Binding Buffer (100 ml)
20 mM Tris-HCl (pH 7.5) --------------------------- 2 ml of 1 M Tris-HCl
800 mM NaCl --------------------------------------- 16 ml of 5 M NaCl
2 mM EDTA ----------------------------------------- 0.4 ml of
0.5 M EDTA
0.2% SDS -------------------------------------------- 2 ml of
10% SDS DEPC-treated H2O ---------------------------------- 79.6 ml
Wash Buffer (100 ml)
10 mM Tris-HCl (pH 7.5) --------------------------- 1 ml of 1 M Tris-HCl
100 mM NaCl --------------------------------------- 2 ml of 5 M NaCl
1 mM EDTA ---------------------------------------- 0.2 ml of 0.5 M EDTA
0.1% SDS ------------------------------------------- 1 ml of 10% SDS DEPC-treated H2O ---------------------------------- 95.8 ml
Elution Buffer (100 ml)
10 mM Tris-HCl (pH 7.5) --------------------------- 1 ml of 1 M Tris-HCl
1 mM EDTA ---------------------------------------- 0.2 ml of 0.5 M EDTA
0.1% SDS ------------------------------------------- 1 ml of 10% SDS DEPC-treated H2O ---------------------------------- 97.8 ml
1. Pour 0.25 g oligo(dT)-cellulose preparation into the pasteur
pipette column with glass wool at the bottom.
2. Equilibrate the column with 10 ml 1 X Binding Buffer.
3. Dilute up to 50 mg total RNA sample to 250 ul with DEPC-treated
H2O.
4. Leave RNA sample at 65oC for 5 minutes.
5. Add 250 ul 2 X Binding Buffer and apply the total RNA sample to the column.
6. Collect the flow-through, leave it at 65oC for 5 minutes and reapply to the column.
7. Wash the column with 5 ml 1 X Binding Buffer.
8. Wash the column with 3 ml Wash buffer.
9. Elute poly A+-mRNA with 3 ml Elution Buffer on ice.
10. Add 0.1 vol of 3 M Sodium acetate (pH 7.0) and 2.5 vol of ice-cold absolute ethanol to precipitate
poly A+-mRNA.
11. An overnight precipitation at -20oC maximizes the precipitation of RNA.
12. Centrifuge at 15,000g for 20 minutes to pellet the RNA.
13. Redissolve the poly A+-mRNA pellet in 50 ul DEPC-treated H2O.
14. Store RNA at -80oC.
15. RNA is stable at -80oC for 6 months.
16. Regenerate oligo(dT)-cellulose as in MATERIALS AND SOLUTIONS.
NOTES
Maximum binding capacity ;
50 mg total RNA / 0.25 g oligo(dT)-cellulose
SDS can be omitted from the 2 X Binding Buffer as it may precipitate
in cold temperature and clog the column. Residual SDS may coprecipitate
with the RNA and interfere with reverse transcription.
KIT INFORMATION
Several kits are available, whereby poly-A+ RNA can be isolated directly from cell or tissue lysates on a small scale. Kits can be divided into two types:
Paramagnetic particles that either contain covalently attached
oligo(dT) or streptavidin. The hybridization step is carried out
in solution between poly-A+ RNA and biotin-tagged oligo(dT), and the hybrids are collected on the streptavidin-coated paramagnetic beads.
Small oligo(dT)-containing cellulose beads that are able to stay
in a fine suspension during hybridization and is pelleted by centrifugation.
REFERENCES
Aviv, H, Leder, Pn (1972) Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose.
P.N.A.S. 69: 1408-1412.
Methods in Molecular Biology. vol. 86 (1998) RNA isolation and
characterization protocols. Ralpley, R and Manning D.L. (eds.),
Humana Press.