Protocol 3: Preparation of mRNA

1. For strains in which you do not expect a general loss of mRNA use 1 mg of RNA (total). If you expect a general loss of mRNA (srb4 ts, rpb1 ts) use 2 or 3 mg total RNA.
   
   
2. Measure OD260 / OD280 of all total RNA preps accurately and in parallel (e.g. by measuring 5 µl of a 5:100 dilution). Accurate determination of the amount of total RNA is essential for correct normalization of results when normalizing with the spiked controls.
   
   
3. Place total RNA with 5 µl of poly-A controls per mg total RNA into each tube.
   
   
4. For 1 mg total RNA: add ODB buffer (Qiagen Oligotex kit) to make 920 µl total volume. Note: Be sure ODB + OLI Buffers are completely dissolved (may need to heat tube a little).
   
   
5. Add 230 µl of OL1 buffer to each tube.
   
   
6. Heat Oligotex bead suspension to 37°C + mix (vortex) immediately before adding 70 µl to each tube. Mix gently by pipetting up and down.
   
   
7. Place tube at 65°C for 3 min. Mix, let sit at room temperature for 10 min with occasional mixing.
   
   
8. Spin 2 min at 14,000 g at room temperature. Aspirate supernatant (leave ~ 50 µl in tube).
   
   
9. Add 600 µl OW1 wash buffer. Resuspend beads by pipetting gently. Spin 2 min at RT and aspirate. Repeat for a total of three washes (1X 600 µl OW1, 2X 600 µl OW2). Remove remainder with pipette after last wash.
   
   
10. Resuspend beads in 175 µl DEPC - dH₂O. Resuspend by pipetting up and down.
    
    
11. Place tube in 65°C waterbath for 1 min. Spin down 1 min at 14,000 g RT. Transfer eluate to fresh tube.
    
    
12. Repeat elution with 175 µl DEPC - dH₂O. Pool eluates.
    
    
13. To get rid of any beads: spin pooled eluates 5 min at 14,000 rpm at RT. Remove 330 µl (know exact amount), transfer to new tube.
    
    
14. Measure OD260 / OD280 of 30 µl of eluate diluted with 270 µl dH₂O.
    
    
15. To remainder add 1/10 volume (30 µl) 3M NaOAc pH 5.2, 1 µl 20 mg/ml glycogen, 675 µl Ethanol (-20°C).
    
    
16. Place 30' at -80°C or overnight at -20°C.
    
    
17. Spin 30' at 14,000 rpm, 4°C. Remove supernatant with pipette.
    
    
18. Wash with 140 µl of 80% Ethanol (-20°C). Spin, remove with pipette. Remove wash completely.
    
    
19. Resuspend pellet with appropriate volume of DEPC - dH₂O to give a final concentration of 0.5 mg/ml.
    
    
20. Snap freeze, store at -80°C.
    
    

PolyA tagged lys, phe, thr, trp and dap T7/T3 IVT expression constructs were obtained from the ATCC (#'s 87482, 87483, 87484, 87485, 87486, respectively). The polyA tagged RNA was generated with NotI digested template DNA and Ambion's Megascript T3 IVT kit according to their instructions. Transcripts were purified using the Qiagen RNeasy kit. An undiluted stock of these controls was prepared by mixing the various transcripts: 133 µg lys, 58.5 µg phe, 24.5 µg dap, 8.8 µg thr, 3.3 µg trp in 225 µl DEPC water. This was aliquoted in 22.5 µl amounts. A diluted stock was made by adding 477.5 µl DEPC water to the undiluted stock and this was again aliquoted. The diluted stock was spiked into the total RNA, 5 µl per mg total RNA. The final amounts of the controls in the total RNA were then: 4 trp, 13.3 thr, 40 dap, 133.3 phe, 400 lys (pmol/mg). By using theses same controls in every experiment all experiments can be normalized to equivalent amounts of total RNA.