In Vitro Transcription Assay (Run-off&nb

Description
With this protocol, transcripts that were initiated from specific genes by RNA polymerases prior to permeabilization can be measured. Instead of a nuclear extract, permeabilized cells are used.

Procedure
A. Permeabilization of Cells

1. Harvest tissue culture cells by trypsinization or scraping into sterile, ice-cold PBS.

2. Count the cell suspension by a cell counter. If clumping is evident (or even suspected), count with a hemacytometer. An accurate cell count is important.

3. Collect cells by centrifugation at 1,000 X g for 5 min at 4°C.

4. Aspire supernatant. Resuspend cell pellet in 10 ml of PBS.

5. Centrifuge at 1,000 X g for 5 min at 4°C.

6. Suspend cell pellet in Permeabilization Buffer to a cell concentration of 108 cells/ml.

7. Check permeabilization by taking 10 ìl of the cell suspension and mixing it with Trypan Blue. Determine the percentage of cell viability by recording the percent of cells that exclude the blue dye.

8. Keep the cell suspension on ice.

9. Add one-one hundredth volume of Lysolecithin. Mix gently but thoroughly.

10. Incubate for 1 min at 4°C.

11. Pellet cells at 800 X g in a table-top centrifuge or equivalent for 3 min at 4°C.

12. Remove the supernatant. Resuspend the cells in 1 ml of Permeabilization Buffer.

13. Pellet the cells at 1,500 rpm in a table-top centrifuge for 3 min at 4°C.

14. Resuspend permeabilized cells in Transcription Buffer to a concentration of 4 X 108 cells/ml.

15. Store cells as 250 ìl aliquots at -70°C.

B. In vitro Transcription Reaction (Run-off)

1. Thaw 1 tube (250 ìl, 1 X 108 cells) of permeabilized cells on ice. Suspend gently.

2. If less than 108 cells are to be transcribed, than take desired volume of cells and dilute to 250 ìl with Transcription Buffer.

3. Add to the cell suspension

1.5 ìl CPK to give a final concentration of 10 ìg/ml

7.6 ìl of Creatine Phosphate to give a final concentration of 10 mM

20 ìl of Combined NTPs to give a final concentration of 220 ìM

25 ìl of [32P] GTP (>3,000 Ci/mmol) (CAUTION! see Hint #1)

The total volume should be 304.1 ìl (see Hint #2).

4. Mix gently and incubate at 30°C for 30 min. Vortex 4 or 5 times during the incubation.

5. Take 1 ìl aliquots at various time points during the incubation, for example, at 0, 10, 20, and 30 min.

6. Use the 1 ìl aliquots to determine the amount of incorporation of label into TCA precipitable counts (see Section F)

7. At the end of the 30 min incubation, centrifuge the samples at 1,500 rpm in a microcentrifuge for 3 min. Discard the supernatant in radioactive waste receptacle.

C. Isolation of RNA

1. Resuspend the cell pellet in 0.5 ml of Guanidinium Thiocyanate Buffer.

2. Add 50 ìl of 2 M Sodium Acetate, pH 4. Mix thoroughly by inversion.

3. Add 0.5 ml of Phenol. Mix thoroughly by inversion.

4. Add 100 ìl of Chloroform:IAA. Mix thoroughly by inversion.

5. Vortex vigorously for 10 sec.

6. Incubate on ice for 15 min.

7. Centrifuge at 10,000 X g for 20 min at 4°C.

8. Transfer the upper aqueous phase containing the RNA to a new microcentrifuge tube. Avoid the white cloudy interface that contains DNA and protein.

9. Add 1 volume of 100% Isopropanol (or 2 volumes of 100% Ethanol).

10. Incubate at -20°C for at least 1 hr to precipitate the RNA.

11. Centrifuge at 10,000 X g for 20 min at 4°C to pellet the RNA.

12. Transfer the pellet with a pipet tip or a sterile Pasteur pipet to a new 1.5 ml microfuge tube.

13. Resuspend the pellet in 0.3 ml of Guanidinium Thiocyanate Buffer. Heating to 65 to 70°C and vortexing will help resuspend the pellet.

14. Add 1 volume of Isopropanol (or 2 volumes of absolute Ethanol).

15. Incubate for 1 hr at -20°C to precipitate the RNA.

16. Centrifuge at maximum speed (13,000 rpm) in a microcentrifuge for 10 min at 4°C to pellet the RNA. Remove the supernatant.

17. Add 1 ml of 75% Ethanol to wash the RNA pellet.

18. Centrifuge at maximum speed (13,000 rpm) in a microcentrifuge for 10 min at 4°C to pellet the RNA.

19. Allow the pellet to air dry. Avoid over-drying the pellet.

20. Resuspend the RNA pellet in DEPC-treated TE or DEPC-treated ddH2O (see Hint #3).

D. Preparation of Slot Blot Membrane for Hybridization (see Hint #4).

1. Add 1 volume of 0.5 N NaOH to the target DNA (see Hint #5). Two ìg of DNA are needed for each slot of the slot blot

2. Incubate for 15 min at 85°C to denature the DNA.

3. Incubate on ice for 5 min.

4. Cut a piece of nitrocellulose to the size of the slot-blot vacuum manifold.

5. Equilibrate the nitrocellulose membrane in 10X SSC.

6. Assemble the slot blot apparatus according to the manufacturer's instructions.

7. To the denatured DNA, add 0.15 volume of 7.5 M Ammonium Acetate and sufficient 10X SSC to dilute the DNA concentration to 10 ng/ìl.

8. Add 200 ìl of the DNA solution to each of the slots of the slot blot apparatus.

9. Apply a vacuum to the dot blot apparatus to draw the DNA solution through the membrane.

10. Rinse each slot with 10X SSC.

11. Bake the nitrocellulose filter at 80°C for 2 hr in a vacuum oven to link the DNA to the membrane.

E. Hybridization of Nitrocellulose Membrane

1. Prehybridize the membrane for 1 hr at 55°C in Hybridization Solution or a hybridization solution of your choice.

2. Add isolated transcribed RNA (from Step #C20) to the Hybridization Solution the membrane is in.

3. Incubate overnight at 55°C to hybridize the transcribed RNA to the DNA on the membrane (see Hint #6).

4. Wash the membrane twice for 15 min each in Wash Buffer 1 at room temperature (see Hint #7)

5. Wash the membrane twice for 15 min each in Wash Buffer 2 at 55°C.

6. Expose the film to membrane for autoradiography.

7. Develop film in 3 to 7 days.

F. TCA Precipitation to Determine Incorporation of [32P] GTP into Nucleic Acid (see Hint #8)

1. Add 1 ìl aliquot of reaction assay to a 13 x 100 mm tube containing 2 ml of Yeast tRNA Solution.

2. Add 1 ml of 10% TCA. Mix.

3. Incubate on ice for 10 min.

4. Wash Whatman glass microfiber GF/C filters with 5 ml of 5% TCA using a vacuum manifold (with the vacuum on).

5. Pour the sample onto the filter.

6. Rinse the tube with 5% TCA and pour the rinse onto the filter.

7. Repeat Step #F6.

8. Wash the filters twice with approximately 5 ml of 5% TCA.

9. Wash the filters twice with 5 ml of 95% Ethanol

10. Turn off the vacuum and remove the filters.

11. Dry the filters thoroughly at room temperature or in a warm (42°C to 100°C) oven.

12. Place filters in scintillation vials.

13. Add Scintillation fluid.

14. Count in a scintillation counter.

Recipes
PBS pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
137 mM NaCl
1.8 mM Potassium Phosphate Monobasic (KH2PO4)

75% (v/v) Ethanol

7.5 M Ammonium Acetate

0.5 M NaOH

Chloroform:IAA 49:1 Chloroform:Isoamyl Alcohol (CAUTION! see Hint #1)

Phenol Store at 4°C in a dark glass bottle
Water-saturated Phenol (CAUTION! see Hint #1)

2 M Sodium Acetate, pH 4

Guanidinium Thiocyanate Buffer 4 M Guanidinium Thiocyanate
Add the 2-Mercaptoethanol just before use.
0.5% Sarkosyl
25 mM Sodium Citrate, pH 7.0
Can be stored for up to 3 months without the 2-Mercaptoethanol
0.1 M 2-Mercaptoethanol

á-Amanitin (1,000X) Store at -20°C
2 mg/ml á-Amanitin
Prepare in ddH2O

Combined NTPs 500 ìl 10 mM UTP
500 ìl 10 mM ATP
Store aliquots at -70°C
500 ìl 10 mM CTP

[32P] GTP Greater than 3,000 Ci/mmol (CAUTION! see Hint #1)

95% (v/v) Ethanol

10 mM UTP Store 100 ìl aliquots at -70°C
Prepare in 10 mM Tris-Cl, pH 7.5
Filter sterilize

10 mM CTP Store 100 ìl aliquots at -70°C
Prepare in 10 mM Tris-Cl, pH 7.5
Filter sterilize

10 mM ATP Store 100 ìl aliquots at -70°C
Prepare in 10 mM Tris-Cl, pH 7.5
Filter sterilize

Yeast tRNA Solution 50 mM EDTA, pH 8.0
1% (w/v) Yeast tRNA

Phosphocreatine Prepare fresh
Prepare in ddH2O
100 mg/ml Phosphocreatine, disodium salt; hydrate

Creatine Phosphokinase (CPK) Prepare in Transcription Buffer
50% (v/v) Glycerol
0.2% (w/v) Creatine Phosphokinase, Type I
Store at -20°C

5% TCA 5% (v/v) TCA Stock

Transcription Buffer 6 mM Magnesium Acetate
20 mM Tris-Cl, pH 8.0
100 mM KCl
Filter Sterilize
Store at 4°C
10 mM Ammonium Chloride
0.3 mM EDTA
Add 2 ìl of 0.5 M DTT per ml of buffer right before use.
10% (v/v) Glycerol

10% TCA 10% (v/v) TCA Stock

0.5 M DTT Store 500 ìl aliquots at -20°C

100% TCA Stock Dissolve in 227 ml ddH2O
500 g Trichloroacetic Acid (TCA)

0.4% Trypan Blue Prepare in PBS
0.4% (w/v) Trypan Blue

Wash Buffer 2 0.1X SSC
0.1% SDS

1% Lysolecithin Store at -20°C
10 mg/ml L-alpha-Lysolecithin, Type I

Wash Buffer 1 0.1% SDS
2X SSC

Permeabilization Buffer 33 mM Ammonium Chloride
150 mM Sucrose
Autoclave
30 mM HEPES, pH 7
Store at 4°C
4.5 mM Magnesium Acetate
7 mM KCl

Denhardt's Solution (100X) 10 g Ficoll 400
10 g Polyvinylpyrrolidone
10 g Bovine Serum Albumin (Fraction V)
Bring final volume to 500 ml with ddH2O
Store at -20°C in aliquots

Hybridization Solution 1% (w/v) SDS
5X SSC
Add DNA just before use.
100 ìg/ml Heat-denatured sheared salmon sperm DNA (or Herring testes DNA)
5X Denhardt's Solution
50% (v/v) Formamide (CAUTION! see Hint #1)

SSC (10X) pH 7.2
0.15 M Sodium Citrate
1.5 M NaCl

95% (v/v) Ethanol

Supplies

Tips
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. To measure the level of RNA polymerase II activity, i.e. hnRNA synthesis, add 1 ìl of alpha-amanitin to a final concentration of 2 ìg/ml.

3. To minimize degradation of RNA by RNAses, wear gloves when handling samples and reagents and change gloves regularly while working. For more information and tips, see "Working with RNA" in the Reference Pages.

4. For each reaction, prepare DNA slots with the DNA(s) of interest and a negative control of plasmid DNA.

5. The in vitro transcribed RNA (from Section B) is detected by hybridization to target DNA.

6. Hybridization up to 72 hr is fine.

7. To decrease background signal, add RNAse A to 1 mg/ml to the Wash Buffer 1.

8. Counts incorporated into nucleic acid are precipitated by TCA. [32P]-GTP is not precipitated.
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In Vitro Transcription Assay (Run-off&nb