Telomeric Repeat Amplification Protocol

Description
This protocol describes a semi-quantitative in vitro assay to detect telomerase activity. Any active telomerase present in a cell extract from detergent-lysed cells will add a variable number of telomeric repeats (TTAGGG) to the 3' end of a substrate oligonucleotide. PCR that incorporates a radiolabeled nucleotide is then used to amplify the extension products. The presence of telomerase in the cell extracts is assessed by the appearance of a ladder of PCR products on a native polyacrylamide gel.

Procedure
A. Preparation of TRAP Reaction Tubes

1. Suspend CX Primer at a concentration of 50 ng/ìl in water with a trace amount of Bromophenol Blue added.

2. Put 2 ìl of CX solution into HotStart 50 PCR tubes (with wax gem on the side of the tube, Molecular Bio-Products).

3. Place tubes in a thermal cycler with a heated top.

4. Run 1 cycle at:

4 min, 75°C

1 min, 85°C

5. Cool at 5 sec/1°C to 20°C.

6. Store at 4°C for up to 1 month.

B. CHAPS Extraction of Cells

1. Combine on ice 1 ml of Lysis Buffer (containing PMSF and 2-Mercaptoethanol).

2. Trypsinize one 100 mm plate of cells. Take 200 ìl to count the number of cells.

3. Centrifuge cells for 5 min at 1,500 rpm in a table-top centrifuge in a 10 ml conical tube.

4. Discard the supernatant, wash the cells in ice-cold PBS, and centrifuge again at 1,500 rpm for 5 min.

5. Discard the supernatant and resuspend the cells at 106 cells/ml in ice-cold PBS.

6. Put 1 ml aliquots of the cell suspension into microcentrifuge tubes.

7. Centrifuge the cells at 10,000 X g for 1 min at 4°C.

8. Resuspend the cells in 20 ìl cold Lysis Buffer (or 20 ìl/104 to 106 cells).

9. Incubate the cell samples on ice for 30 min (see Hint #1).

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

11. Collect the supernatant, flash freeze it in liquid nitrogen, and store at -70°C.

12. Extracts may be freeze-thawed up to 5 times (see Hint #2). Expect 5 to 10 mg/ml protein.

C. TRAP Reaction

1. Number reaction tubes containing the CX primer under wax. Always run a tube containing RNase in the extract as a negative control for the telomerase assay and an ddH2O blank as a negative control for the PCR reaction.

2. Add 2 ìl DEPC-treated ddH2O to the experimental tubes, which will not contain RNase.

3. Add 2 ìl of CHAPS extract prepared in Section B to each tube (or water to the control tubes).

4. Add 2 ìl of RNase to the negative control tubes, centrifuge the tubes briefly, and let them incubate for 10 min at room temperature.

5. Change gloves to avoid contaminating the experimental samples with RNase.

6. Make up enough of the following mix for the number of reactions to be done plus 10% volume.

Per single reaction set up:

25 ìl of 2X TRAP Buffer

17 ìl of DEPC-treated ddH2O

1.0 ìl of 10 mM dNTPs Stock

2.0 ìl of 50 ng/ìl TS primer

0.1 mg/ml of BSA

7. Mix by inverting, then add:

Magnesium Chloride to a final concentration of 1.54 mM

0.1 ìg of TS Primer

0.1 ìg of CX Primer

2 Units of Taq DNA Polymerase mixed with an equal amount of TaqStart Antibody as per directions supplied with TaqStart, Clonetech.

2 ìl of CHAPS detergent

0.2 ìl of á-[32P]-dCTP 10 ìCi/ìl, 3,000 Ci/mMol (Amersham) (CAUTION! see Hint #3)

46.0 ìl of 200 ìM combined dNTPs (see Hint #4)

8. Mix again by inverting and centrifuge briefly to collect all the liquid at the bottom of the tube.

9. Add 46 ìl of the mix to each tube prepared in Steps #C1 to #C4, mix and centrifuge briefly. Do the RNase control tubes last.

10. Incubate tubes at room temperature for 10 min (while telomerase extension occurs).

11. Put tubes into PCR thermal cycler preheated to 90°C and incubate for 1 min (see Hint #5).

12. Immediately start the following cycle program:

94°C, 30 sec

50°C, 30 sec

70°C, 1.5 min

31 cycles

D. Electrophoresis of Samples on Native Polyacrylamide Gel

1. Pour a 15% Polyacrylamide Gel. Use 15 cm glass plates with 0.4 mm spacers and comb from a sequencing apparatus cut to fit the smaller 15 cm plates.

2. Mix 8 ìl reaction product with 2 ìl Loading Dye and load on the gel.

3. Run the gel at 700 V for approximately 2 hours in 1X TBE.

4. Fix the gel by immersing it in 7% Acetic Acid for 5 min.

5. Rinse the gel in ddH2O briefly.

6. Dry the gel on Whatman 3MM paper.

7. Expose to film for about 12 hours on Bio-max film (Kodak).

8. For quantitation, compare the signal intensity of the bands of the ladder to the signal obtained from an internal control band.

Recipes
0.25 M EGTA Add NaOH while stirring until it goes into solution
Store at 4°C
Prepare in DEPC-treated ddH2O
Filter

1 M Tris-HCl, pH 8.3 When checking pH on pH meter, don't put the probe in the RNase-free buffer
Store at 4°C
Filter
Prepare in DEPC-treated ddH2O (see Hint #8)

1 M Tris-HCl, pH 7.5 When checking pH on pH meter, don't put the probe in the RNase-free buffer
Store at 4°C
Prepare in DEPC-treated ddH2O
Filter

10 mM dNTPs Stock (see Hint #4) 2.5 mM dGTP
2.5 mM dCTP
2.5 mM dTTP
2.5 mM dATP

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

7% (v/v) Acetic Acid

TBE 2 mM EDTA, pH 8.0
89 mM Tris
89 mM Boric Acid

15% Polyacrylamide Gel 6 ml of ddH2O
Makes enough for one 0.4 mm gel
105 ìl of 10% (w/v) Ammonium Persulfate (not less than 3 weeks old)
5.25 ìl of TEMED
7.5 ml of 30% Acrylamide (29:1 Acrylamide: Bisacrylamide) (CAUTION! see Hint #7)
1.5 ml of TBE

Loading Dye for DNA 0.25% (w/v) Xylene Cyanol
0.25% (w/v) Bromophenol Blue
30% (v/v) Glycerol

TE 10 mM Tris
PH 8.0
1 mM EDTA

TS Primer Prepare in TE
500 ng/ìl
Primer #2: 5'-AATCCGTCGAGCAGAGTT-3'

CX Primer Prepare in TE
Primer #1: 5'-(CCCTTA)3 CCCTAA-3'
500 ng/ìl

TRAP Buffer (2X) 1.5 mM MgCl2
Store 1 ml aliquots at -20°C
Filter sterilize
1 mM EGTA
63 mM KCl
Prepare in DEPC-treated ddH2O
0.005% (v/v) Tween 20
20 mM Tris HCl pH 8.3
0.1 mg/ml BSA

Lysis Buffer Prepare in DEPC-Treated ddH2O
1 mM MgCl2
Filter sterilize
10 mM Tris HCl, pH 7.5
1 mM EGTA
Store 1 ml aliquots at 4°C
0.5% (w/v) CHAPS Detergent
10% (v/v) Glycerol
Add 1 ìl of 0.1 M PMSF and 0.3 ìl of 2-Mercaptoethanol just prior to use (CAUTION! see Hint #7)

0.1 M PMSF Divide into 1 ml aliquots and store at -20°C
Add 0.174 g PMSF to 10 ml Isopropanol (CAUTION! see Hint #7)

1 M KCl Store at room temperature
Autoclave
Prepare in DEPC-treated ddH2O

1 M Magnesium Chloride Store at room temperature
Autoclave
Prepare in DEPC-treated ddH2O

Supplies

Tips
1. This allows the telomerase to leak out of the cells.

2. The extracts of the contributor of this protocol survived a freezer meltdown.

3. CAUTION! When using radioisotopes, always follow your institution's policies and regulations regarding the use and disposal of radioactive materials. Be sure to use proper shielding and take all necessary precautions to protect yourself from undue exposure to radioactivity.

4. The contributors of this protocol use DNA Polymerization Mix (Pharmacia) as their source of combined dNTPs Stock.

5. You might want to set the thermal cycler for 2 to 3 min because it may take while to load the tubes.

6. To minimize degradation of RNA by RNases, wear gloves when handling samples and reagents and change gloves regularly while working. Treat water and solutions with DEPC* (diethyl pyrocarbonate) to inactive RNases and use solutions prepared with RNase-free ddH2O and equipment. Use sterile plasticware or glassware that have been baked at 150°C in dry heat for at least 2 hours. Autoclaving solutions and vessels are not sufficient to remove all RNase activity. Do not use spatulas to measure out chemicals used for RNase-free solutions. Reserve RNase-free solutions, reagents, and consumables, such as pipette tips, only for RNA work.

Some researchers find that following all of the above measures is not necessary for good quality RNA preps. You must determine for your own work how conservative to be in eliminating sources of contaminating RNases.

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

8. DEPC also reacts with amines and so cannot be used with Tris buffers before it is inactivated. Make up the Tris solution using DEPC-treated ddH2O
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Telomeric Repeat Amplification Protocol