Nuclear runon assay

1 Defrost isolated macrophage nuclei.

2 Defrost [MP]UTP using appropriate radiosafety protocols,

3 Make up sufficient 5 x run-on buffer in sterile microcentrifuge tube to give 60 jjlI per sample.

4 Bring 150 of isolated nuclei preparation to 200 with freezing buffer in a 0.5 ml microcentrifuge tube, preparing a separate sample for each DNA to be assessed,

5 Add 60 |xl of run-on buffer to each sample.

6 Add 100 mCi of labelled UTP to each sample, in a final volume of 30 p,l.

7 Cap tube and vortex briefly to mix.

8 Incubate at 30°C for 30 min.

9 At the end of this incubation, add 15 jil of RNase-free DNase I.

10 Incubate again at 30 °C for 5 min.

11 Add 36 m.1 of 10 x SET buffer and 10 jri of proteinase K (10 mg/ml stock).

12 Heat tubes transiently to 65 °C, if needed, to redissolve SDS.

14 Add 360 jri (approximately equal volume) of PCI at completion of incubation and vortex to mix.

15 Spin 5 min in microcentrifuge at 12 000 gat room temperature to form a gradient.

16 Remove the upper (aqueous) phase containing the RNA using a sterile Pasteur pipette (avoid touching interface or lower phase).

17 Transfer the RNA to a fresh microcentrifuge tube and hold on ice.

18 Add 100 fil of 1 x SET buffer to the original tubes.

19 Re-extract the interface and Lower gradient phases as in steps 14 and 15.

20 Remove the aqueous phase from the second gradient using a sterile Pasteur pipette as in step 16.

21 Pool the aqueous phases from the two extractions in a single tube.

22 Add 135 jjlI of 10 M ammonium acetate and 595 ^il of isopropyl alcohol to each tube.

24 Remove tubes from the freezer and thaw on ice.

25 Spin tubes for 10 min at 4 °C, 12 000 g in a microcentrifuge to pellet the RNA.

26 Remove 'hot' supernatant at the completion of the centrifugation and collect for proper radioactive disposal,

27 Resuspend the RNA pellet in 180 of 1 x STE buffer.

29 Incubate on ice for 10 min,

31 Precipitate with 880 of ethanol overnight at -20°C (or on dry ice for lOmin).

32 Pellet the precipitated RNA by centrifugation for 20 min at 4°C, 12 000g.

33 Resuspend pellet in 1 ml of run-on hybridization buffer.

34 Count 10 jj.1 of the resuspended RNA in scintillation fluid using a glass scintillation vial.

35 Add 5-10 x 106 c.p.m. of radioactivity per membrane in 2-5 ml.d

36 Incubate membranes at 65#°C for 36-48 h with frequent mixing.

37 At the completion of the incubation, rinse the membranes briefly in a 1:1 mixture of 0.1 X SSC and 0.1% SDS at room temperature and remove the wash fluid (fluid from steps 37 and 38 should be disposed of as radioactive waste).

38 Wash membranes twice, for 30 min each, at 65 °C in a 1:1 mature of 0.1 x SSC plus 0.1% SDS.

39 Expose membranes to radiographic film for appropriate times and develop for aut oradiography.

a The phenol used in this procedure should be redistilled from commercially available products as follows. Melt phenol at 65°C and equilibrate. Place in a separatory funnel with equal volume of 0.1 M Tris pH 8.0 and mix by shaking. Let stand for 10 min until the mixture separates—the phenol is in the lower (aqueous) phase. Retrieve phenol and re-extract a total of four times, or until the pH of the aqueous phase is approx. pH 7.6. Extracted phenol should be used immediately or frozen at -20 °C until use. Appropriate safety precautions should be observed during phenol extraction, including protective eyewear.

b Pre-hybridized membranes can only be used once, and the radiolabeled RNA is used in great excess; thus, it is most efficient to spot as many genes as possible on each slot blot. c Generally, membranes are prepared over two to three days, using DNA that has been purified over the course of a few weeks (e.g. for five to ten genes). If needed, membranes can be cut with a razor blade prior to hybridization.

d it is generally advisable to keep the volume of hybridization buffer as small as possible, to ensure the greatest interaction of the fluid with the membrane during incubation.

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