Deadenylation Assay

Day 1

1. Synthesis of capped RNA transcripts

  • In vitro transcription using MaxiScript Transcription Kit (Ambion)
Reagent 1x
RNase-free H2O 2,25 ul
Linearized DNA template (500 ng)
1 mM GTP 0.5 ul
10 mM ATP 0.5 ul
10 mM CTP 0.5 ul
200 uM UTP 0.75 ul
10 mM cap analog 0.5 ul
10x transcription buffer 1 ul
800 Ci/mmol, 20 mCi/ml α32P-UTP 3 ul
T7 enzyme mix 1 ul
Total volume 10 ul

Incubate reaction at 37°C, 1 hr

2. Removal of template DNA

i.  Add 1 ul DNase Turbo (Ambion), quick spin
ii.  Incubate at 37°C, 15 min

3. Inactivate DNase by adding 1 ul 0.5 M EDTA pH 8

4. Adjust rxn to 60 ul (add 48 ul H2O)

5. Determine radiolabel UTP incorporation:

i.  Take 1 ul (from 60 ul transcription rxn) and add to 5 ml scintillation cocktail à “before”
ii.  Take 1 ul from Step 6vi and add to 5 ml scintillation cocktail à “after”

6. Removal of free nucleotides

  • Using Sephadex G-25 Mini Quick Spin RNA Columns (Roche) & NH4OH/EtOH pptn

i.  Resuspend column by inverting it
ii.  Remove top cap, snap off bottom tip
iii.  Place column in 1.5 ml tube and © at 1000xg, 1 min, RT à use kimwipe to remove excess liquid at the bottom of the column
iv.  Place prepared column in new 1.5 ml tube
v.  Load rxn from step 4 (after completing Step 5i) to center of column
vi.  © 1000xg, 4 min, RT
vii.  Add 10 ul 5-8M NH4OH and 250 ul 100% cold EtOH
viii.  Ppt RNA at -20°C, 1 hr
ix.  © Vmax, 20 min, 4°C
x.  Remove supt, and add 125 ul 70% cold EtOH
xi.  © V­max, 5 min, 4°C
xii.  Remove supt. Air dry pellet for 10 mins.
xiii.  Resuspend pellet in RNase-free H2O (1 ng/ul RNA based on calculations obtained from Step 5).

Day 2

7. Deadenylation assay

Reagent 1x
Suppl. extract 9.136
RNA (1 ng/ul) 1
H2O 2.364
Rxn volume 12.5 ul

Incubate at 17°C

  • Set up master mix reaction for number reactions needed.
  • 1x reaction per time point.
  • Transfer 12.5 ul to a new microtube at each time point and add 1 ml QIAzol. Mix and leave on ice until time course is complete.

8. RNA extraction

i.  Once time course is complete, add 200 ul CHCl3 to QIAzol-treated samples and vortex for 15 sec. Incubate at RT, 5 min.
ii.  © 12,000xg, 10 min, 4°C.
iii.  Collect 550 ul aqueous phase and transfer to new tube containing 1 ul glycoblue.
iv.  Add 550 ul isopropanol, mix by inverting tubes, and allow RNA to ppt O/N or 1 hr at -20°C.
v.  © 12,000xg, 10 min, 4°C.
vi.  Remove supt and wash pellet with 1 ml cold 70% EtOH, mix by inverting tubes.
vii.  © 7,500xg, 5 min, 4°C.
viii.  Remove supt and do 1 more quick spin to remove as much supt as possible.
ix.  Dry pellet and resuspend in 50 ul 1x Gel loading buffer II (Ambion).
x.  Mix by flicking tube, then quick spin.

Day 3

10. RNA analysis on denaturing gel

i.  Heat samples at 95°C for 2 min.
ii.  Place samples on ice, 5 min.
iii.  Load samples (5 ul) on 4% urea-acrylamide denaturing gel
iv.  Run gels at 165V in 1x TBE, 4h30 (for 1.5 kb RNA)
v.  Dry gels for 2hrs.
vi.  Expose O/N on an imaging plate at RT and Phosphorimager next day.

Source: Wu et al. Mol Cell 2010