The amount of poly(A) in an RNA sample can be determined by digestion of RNA:3H-poly(U) hybrids with RNase A and DNase I. Only that poly(U) annealed to RNA (poly(A)) will remain in TCA precipitable form. This procedure can be used to assay poly(A) in RNA samples from eukaryotic cells (nuclear, cytosolic, polysomal, mito, plastid, total, etc), prokaryotic cells, or archaebacterial cells. RNA from prokaryotes and archaebacteria should be extracted by the SDS-protease K method.
Of course, strict RNase-free technique should be used at all times. WEAR GLOVES, and use only autoclaved or DEPC-treated glassware and buffers. Use high-purity RNase A and RNase-free DNase I. Be sure that the sample is not exposed to TCA for more than 5 min before filtration, as poly(U) is labile in TCA.
- Materials
- Hybridization buffer
| 10mM |
Tris-HCl, pH7.6 |
| 200mM |
NaCl |
| 0.2% |
SDS |
- 200mM KCL + 5mM MgCl2
- 0.13mg/ml RNase A (DNase-free) in HB
- 1.65mg/ml DNase I (RNase-free) in HB
- 2mg/ml sonicated salmon sperm DNA
- 2.5% and 5% TCA
- 3H-poly(U)
Amersham or Miles labs. The higher the specific activity, the more accurate the results.
- Poly(A)
At concentrations of 1000, 100, 10, 1, & 0.1ug/ml
- Methods
To produce a standard curve to compare the experimental data to, first carry out the assay using synthetic poly(A) in place of RNA.
- EtOH precipitate the RNA sample twice, and redisslove the RNA at a concentration of about 1ug/ul in HB.
- Dilute the 3H-poly(U) in 50% EtOH to a concentration of 0.05uCi/ml, and dispense 50ul aliquots into eppendorf tubes. Dry in a rotovac and store at -20 until use.
- Add decreasing amounts of the RNA solution to reaction vials, and add HB to a final volume of 50ul. Vortex. Store at frozen, and procede with the assay with groups of two vials.
- Incubate at 25C for 15 min., and add 50ul 200mM KCl + 5mM MgCl2.
- Mix & incubate at 4C for 20 min. Pellet the SDS-K+ for 2 min. at 4C.
- Transfer the sup to fresh tubes containing 2ul @ 0.13mg/ml RNase A and 1.6mg/ml DNase I, and incubate at 25C for 30 min.
- Transfer to ice, and add 10ul 2mg/ml sssDNA, 115ul 5% TCA and 1ml 2.5% TCA.
- Vortex and filter the solutions within 5 min. Wash each filter with 5ml 2.5% TCA. Dry the filters under a heat lamp, and add them to scintillation vials containing 5-10ml scintillation fluid, and count.
- From the acquired data, plot CPM verses ug RNA. At low RNA concentrations, the curve should be linear. From the poly(A) standard curve, calculate the protected CPM corresponding to each ng of poly(A), and use this value to determine the ng poly(A) per ug RNA in the experimental sample.