Overview

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.

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℃.

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

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

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

7. Check permeabilization by taking 10 μl 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℃.

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

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℃.

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

15. Store cells as 250 μl aliquots at -70℃.

B. In vitro Transcription Reaction (Run-off)

1. Thaw 1 tube (250 μl, 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 μl with Transcription Buffer.

3. Add to the cell suspension

1.5 μl CPK to give a final concentration of 10 μg/ml

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

20 μl of Combined NTPs to give a final concentration of 220 μM

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

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

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

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

6. Use the 1 μl 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.