Poly(A)-containing RNA in an RNA population can be sized by annealing total RNA to 3H-poly(U), followed by sizing by sucrose rate zonal ultracentrifugation. The following technique can be used to rapidly determine the size range of poly(A)+ RNA from any RNA isolate of at least 200ug. Note that denaturing gradients, such as those made in formamide, cannot be used since this would separate the 3H-poly(U) from the poly(A)+ RNA. Therefore, poly(A)+ RNA sizes obtained by this method should only be considered estimates, since the internal rRNAs used to interpolate S values for the poly(A)+ RNA will not migrate solely on the basis of MW, but will be influenced by secondary structure.
Naturally, strict RNase-free technique must be used. Autoclave all glassware and solutions (except sucrose) before use, and use virgin plasticware (including the tubing for collecting fractions!). Pretreat the centrifuge tubes and gradient maker with 0.3% DEPC for 2 hr at room temperature. DO NOT AUTOCLAVE NITROCELLULOSE CENTRIFUGE TUBES!
- Materials
- 50% sucrose
500 grams sucrose plus 500ml ddH2O. Use RNase-free sucrose only. Sol'n is 50% w/w
- 5% sucrose in VAN
|
|
Final Conc. |
| 50ml |
50% sucrose |
5% |
| 50ml |
10X VAN |
1X |
| 400ml |
ddH2O |
50ml |
- 20% sucrose in VAN
|
|
Final Conc. |
| 200ml |
50% sucrose |
20% |
| 50ml |
10X VAN |
1X |
| 250ml |
ddH2O |
50ml |
- 10X Gradient buffer (VAN) (per 300ml)
|
|
Final Conc. |
| 26.31 grams |
NaCl |
150mM |
| 20.40 grams |
Na acetate-3H2O |
50mM |
Adjust to pH 5.1with HOAc
- RNA sample buffer (per 100ml)
| 0.29 grams |
NaCl |
| 0.68 grams |
Na acetate |
Adjust to pH 5.1with glacial HOAc
- 3H-poly(U)
Miles laboratories #57-350. Supplied as 10uCi in 440ul 50% EtOH - 56uCi/A260 unit. Equals 22.7 nCi and 16ng poly(U) per ul
- Gradient preparation:
Prepare two 17ml 5-20% linear gradients in SW28.1 tubes using 5% and 20% VAN buffered sucrose and a gradient maker. Premade gradients should be loaded and run immediately (within hours). Gradients can also be made by carefully layering 4.25ml layers of 20, 15, 10, then 5% TEN buffered sucrose on top of each other in the centrifuge tube. The handmade gradients should then be carefully incubated for overnight at RT to allow the gradient to smooth out by diffusion.
- Sample preparation:
- EtOH precipitate 100-500ug total or polysomal RNA. Pellet in a microfuge 15 min.
- Dissolve the RNA in 50ul sample buffer, then add 1ul 3H-poly(U).
- Incubate at 45C for 10 min., then quickly quench on ice. Add 300ul ice cold sample buffer and carefully layer onto a gradient.
- Ultracentrifugation:
- Run the gradients with slow acceleration (setting of 1-2), brake off, 27KRPM, for 19 hr at 4C. When the rotor coasts to a stop, the w2t should be about 5.5 X 10~11 rad2/sec.
- Fractionation:
- The gradients should be fractionated as soon as possible, one after the other. Fill the tubing with 20% sucrose, to prevent gradient-stirring bubbles, and clamp off the tubing. Puncture the gradient tube and unload the gradients at 1-2 sec/drop (by gravity flow), collecting 10 drop fractions. Refill the tubing with 20% sucrose, and go to the next gradient.
- Analysis:
- Read the A260 of each fraction using a microcuvette, then add 300ul of each fraction to separate scintillation vials containing 5ml Aqueous scintillation fluid and count 3H CPM (5 min counts are sufficient). Graph A260 and CPM verses fraction #, and calculate the S value range of the 3H CPM (representing poly(A)+ RNA) by comparision to the rRNA A260 peaks.