1

520nm Emission

520nm Emission

Excite 495nm

Excite 495nm

Fluorescent Products _>.

Fig. 1. Principle of Amplifluor primer system. (Please see Companion CD for a color version of this figure.)

obtained from Sigma except for CHAPS (Pierce). The buffer is stable when stored

2. 10X TRAP buffer: 200 mM Tris-HCl, pH 8.3, 18 mM MgCl2, 630 mM KCl, 0.5% Tween-20, 10 mM EGTA, and 1.0 mg/mL bovine serum albumin (BSA). Prepare a buffer from stock solutions of 1 M Tris-HCl, pH 8.3, and 1 M MgCl2, and solid KCl and EGTA. Mix and dissolve completely each component, filtrate through 0.45-|im filter, autoclave, cool to room temperature, and add Tween-20 (10% purified Tween-20 solution can be obtained from Pierce) and molecular biology grade BSA (nonacetylated and DNase-, RNase-, and proteinase-free: available from Sigma or Ambion). Store at 2-8°C.

3. 50X dNTP mix (dG, dA, dT, and dCTP). Prepare a stock at 2.5 mM each in a buffer containing 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA (T10Ea1).

4. ET primers:

RPm4 primer: 5'-FAM-ACGCAATGTATGCGT(dabsyl)GGCTTACCCTT ACCCTT ACCCTAACC-3'. Make 50X stock at 7 |M in T10E01 (see Note 1). The RPm4 consists of a 3'-end sequence complementary to the target sequence (telomeric repeats) and a 5'-end sequence which forms a stable 15-nt long intramolecular hairpin structure. The 5'-end of the RPm4 was labeled with FAM (donor) by using FAM modifier amidite (Glen Research) in the last step of the chemical synthesis. The dabsyl (4-[4'-dimethylamino-phenylazo] benzene sul-fonic acid: acceptor), a nonfluorescent chromophore, was linked to T containing a C6 amino group, which is complementary to the 5'-end A when the primer is hairpin structure. The dabsyl moiety was incorporated into the Amplifluor oligo-mer during synthesis utilizing dabsyl-dT phosphoramidite (Chemicon International). The RPm4 was purified by C-18 reverse phase high-performance liquid chromatography (HPLC).

K2m3 primer: 5,-TR-AGGACGTAGAGTCGTCCT(dabsyl)TGGTCAGAT CAGTT CACATC-3'. Make 50X stock at 5 |M in T10E01 (see Note 1).

The 5'-end of K2m3 form 18 nt-long intramolecular hairpin structure similar to RPm4. The sequence of the 3'-end 20 nt is complementary to 3'-end of T42, the template for the internal control (see below). The dabsyl moiety was incorporated during the synthesis as described for RPm4. The 5'-end of K2m3 was labeled with monomethoxytrityl-C6-amine modifier (Glen Research) during the chemical synthesis. After purification by C-18 reverse phase HPLC, the 5 amine was labeled with Texas Red succinimidyl ester (Molecular Probe) followed by C-18 HPLC purification.

5. TS primer. 5' AAT CCG TCG AGC AGA GTT 3' purification by HPLC is recommended. Make 50X stock at 8 |M in T10E01.

6. K4 primer. 5' GAA AGT CAT AGC TAC AGA 3'. Post synthetic purification is not required for K4 primer. Make 50X stock at 5 |M in T10E01.

7. T42 (template for amplification of internal control). 5' GAAAGT CAT AGC TAC AGA TGT GAA CTG ATC TGA CCA 3'. Make 50X stock at 10 amol/|L (see Note 2).

8. TSR8. 5' AAT CCG TCG AGC AGA GTT AGG GTT AGG GTT AGG GTT AGG GTT AGG GTT AGG GTT AGG GTT AG 3'. TSR8 is an oligonucleotide with a sequence identical to the TS primer extended product with eight telomeric repeats AG(GGTTAG)7. This control serves as a standard for estimating the amount of TS primers with telomeric repeats extended by telomerase in a given extract. Make a working stock at 0.2 amol/|L (see Note 2).

9. Taq polymerase: use endonuclease activity-free and antibody-treated "Hot-start" Taq polymerase. The author used Patinum Taq polymerase available from Invitrogen. Chemically modified "Hot-start" Taq polymerases are not recommended as they are not compatible with Amplifluor chemistry.

10. Telomerase-positive control cell pellet: prepare a cell pellet from exponentially growing cultured cells as a telomerase-positive control (see Note 3).

11. Reagents for determination of protein concentration.

12. RNase Inhibitor.

2.2. Equipment

1. Optically clear PCR tubes and caps for PCR amplification and real-time detection: optical tubes (Perkin-Elmer Micro Amp, cat. no. N801-0933) and optical caps (Perkin-Elmer Micro Amp, cat. no. N801-0935b).

2. ABI PRISM® 7700 DNA Sequence Detector.

3. If analyzing tissues, homogenization equipment as described in Subheading 3.1., step 3b.

3. Methods

3.1. Preparation of Telomerase Extract

1. Pellet the cells or tissue, wash once with phosphate-buffered saline (PBS), repellet, and carefully remove all PBS. After removal of PBS, the cells or tissue pellet can be stored at -85 to -75°C or kept on dry ice. Telomerase in frozen cells or tissues is stable for at least 2 yr at -85 to -75°C. When thawed for extraction, the cells or tissue should be resuspended immediately in CHAPS lysis buffer.

2. For cells, resuspend the cell pellet in 200 ||L of CHAPS lysis buffer/105-106 cells. Also use 200 |L of CHAPS lysis buffer for the preparation of telomerase-positive control cell extract.

3. For tissues (see Note 4), prepare the extract according to one of the methods described next. Use 200 |L of CHAPS lysis buffer/40-100 mg of tissue.

a. Soft tissues: homogenization with motorized disposable pestle (VWR, cat. no. KT749520-0000, KT749540-0000). Mince tissue sample with a sterile blade until a smooth consistency is reached. Transfer the sample to a sterile 1.5-mL microcentrifuge tube, and add CHAPS lysis buffer. Keep sample on ice and homogenize with a motorized pestle (approx 10 s) until uniform consistency is achieved.

b. Connective tissues: freezing and grinding. Place tissue sample in a sterile mortar and freeze by adding liquid nitrogen. Pulverize sample by grinding with a matching pestle. Transfer thawed sample to a sterile 1.5-mL microcentrifuge tube, and resuspend in an appropriate amount of CHAPS lysis buffer.

c. Connective tissues: mechanical homogenizer. Mix tissue sample with an appropriate volume of CHAPS lysis buffer in a sterile 1.5-mL microcentrifuge tube placed on ice. Homogenize with a mechanical homogenizer (e.g., PowerGen Model 35 Homogenizer, Fisher, cat. no. 15-338-35H) until a uniform consistency is achieved (approx 5 s). It is critical to keep the sample on ice during homogenization to prevent heat accumulation.

4. Incubate the suspension on ice for 30 min.

5. Spin the sample in a microcentrifuge at 12,000g for 20 min at 4°C.

6. Transfer the supernatant into a fresh tube and determine the protein concentration of the sample extracts and dilute with CHAPS lysis buffer to obtain protein concentration recommended next (see Note 5).

7. Aliquot and quick-freeze the remaining extract on dry ice, and store at -85 to -75°C. The extract is stable for at least 12 mo at -85 to -75°C (see Note 6).

3.2. TRAP Assay on ABI 7700 Sequence Detector

3.2.1. Controls

For a valid analysis of the results, appropriate controls are to be included in every assay.

For each sample:

a. Heat inactivation control: telomerase is a heat-sensitive enzyme. As a negative control, every sample extract to be evaluated should also be tested for heat sensitivity. Thus, analysis of each sample consists of two assays: one with a test extract and one with a heat-treated test extract. Heat treat 10 |L of each sample by incubating at 85°C for 10 min before the TRAP assay to inactivate telomerase.

For each set of TRAP assays:

a. Telomerase quantitation controls: perform the TRAP assay using serial dilutions of TSR8 (control template) instead of sample extract to generate a standard curve. Prepare 1:5 serial dilutions of the TSR8 stock (0.2 amol/|L) with CHAPS lysis buffer to obtain concentrations of 0.04, 0.008, 0.0016, and 0.00032 amol/|L. Perform five assays using 2 ||L of each TSR8 dilution including the 0.2-amol/|L stock. The diluted TSR8 can be stored at -20°C for at least 4 wk.

b. Telomerase positive extract control: make a telomerase-positive cell extract using 200 | L of CHAPS lysis buffer and the cultured cell pellet (106 cells). Aliquot the lysate in microcentrifuge tubes and store at -85 to -75°C. Dilute the stock aliquots 1:20 with CHAPS lysis buffer before use and dispense 2 |L per TRAP assay (2 |L = 500 cells). Run one positive control reaction for each set of assays.

c. No target control: perform a TRAP assay with 2 |L CHAPS lysis buffer substituted for the cell/tissue extract. If the assay worked optimally, only the positive CT in the ROX window that corresponds to the internal control amplification is detected in this control. The amplification in the FAM window indicates either: (1) the presence of primer-dimer PCR artifacts owing to suboptimal PCR conditions; (2) the presence of PCR contamination (amplified TRAP products) carried over from another assay; and/or (3) the contamination of an assay component with the telomerase positive cell extract. Run three no target control reactions for each set of assays.

d. PCR amplification control: (internal control is included in each assay by default). An important feature of the assay described here is the inclusion of an internal standard in every reaction. Many cell/tissue extracts contain inhibitors of Taq polymerase, and, thus, give potentially false-negative results. To distinguish this from other problems, the reaction mix contains the primers and template (Texas Red-labeled K2 Amplifluor primer, unlabeled K4 primer, and the T42 template) for amplification of 56-nt internal control, which is detected in the ROX window of the instrument. The threshold cycle of the internal control is used to monitor PCR efficiency and presence of PCR inhibition. (For more details, see Subheading 3.4.)

The programing described here is focused on the procedures specific to the two-color TRAP assay. For more details about the general procedure of the 7700, refer to the operation manual and user bulletins available from ABI.

3.2.2. Setting Up and Programming of the PRISM 7700

1. Open Sequence Detector v1.6.3 software.

2. Creating plate documents.

Go to [File] for Pull Down Menu. Click Open New Plate. Plate type: Single Reporter, instrument: 7700 Sequence Detector, run: Real Time, click OK. New plate appears on the screen.

Configuring the FAM Dye Layer: Dye Layer Window is FAM. Go to Sample Type pop-up menu, choose Sample Type Set Up. Quencher: OFF Click OK. Identify the wells for standards (STD), no template control (NTC), and unknown (UNKN). For duplicate reaction, give same name in Replicate window. Assign the concentration of the standard TSR8 by clicking the wells: and type the quantity of the TSR8 in TPG unit in the Quantity window. 1 TPG unit = 0.001 amol of TSR8.

Configuring ROX Dye Layer: press Dye Layer window, choose ROX. Go to Sample Type pop-up menu, click Sample Type Set Up. Highlight IPC+(Internal Positive), click ROX in the reporter window. The Quencher dialogue box shows check mark with None selection. Leave as is. Click OK. While the Dye Layer window is ROX, highlight all the wells where reaction tubes are present.

3. Set thermal cycling conditions.

Click Thermal Cycler Conditions window from the Plate document. Click individual time and temperature text fields and enter the following settings: Stage 1: 30°C/30 min. Stage 2: 95°C/2 min. Stage 3: 94°C/15 s, 55°C/60 s, 40 cycles. Set volume to 50 |L.

4. Identify the stage for data collection.

Click Show Data Collection window. Deselect 30, 95, and 94°C measuring by clicking the logo in the field and leave the logo of stage 3 step 2 (55°C annealing and extension step), click OK.

5. Set analysis conditions.

On the plate document, click Data Collection to toggle to Data Analysis. In the [Instrument] pull down menu, choose Diagnostics, and then choose Advanced Option. In the Advanced Options dialog box. Deselect Use Spectral Compensation for Real Time by checking the box. Deselect Reference by checking the dialog box. The "ROX" sign automatically switches to None by the deselection. Click OK. Ignore the warning and click OK. Go to [Analysis] pull down menu and click Options. In the Options dialog box, choose data collection to be at step 2 of stage 3.

6. Save the settings.

Go to the [File] pull down menu, press Save. Choose the file to be stored and type the name of the experiment in the box. Click Save, click Data collection window. Press Run at the upper center of the plate.

3.2.3. TRAP Assay-Amplification and Detection 1. Assay set up.

a. Prepare a "Master Mix" for the assay (50 |L/reaction) by mixing stock reagents listed next in a sterile tube. 10X TRAP buffer, 50X TS primer, 50X RPm4 primer, 50X K2m3 primer, 50X K4 primer, 50X T42 template, 50X dNTP mix, "Hot-Start" Taq Polymerase (1 U/reaction), and H2O quantity sufficient to 48 | L/reaction.

Typically, 3n + 9 reactions are necessary for analysis of n number of sample extracts (duplicate assays and one heat-inactivated control for each experimental sample and other controls). Prepare a master mix, sufficient for 3n + 10 reactions considering pipetting variances.

b. Aliquot 48 ||L of the Mix containing Taq polymerase into 3n + 9 DNase- and RNase-free optically clear PCR tubes (see Note 7).

c. Add 2 |L of test extracts, heat-inactivated extracts, or controls into each tube.

2. PCR amplification: place tubes in the thermocycler block of the Prism 7700.

Start the operation by clicking Run on plate document.

3.2.4. Data Analysis

1. Go to [Analysis] pull down menu and select Options. In the Options dialog box, confirm that analysis uses data collected at stage 2 of extension step 2.

2. In the [Analysis] pull down menu, select Analyze. In the Amplification plot screen. Click OK. In the experiment report screen, click OK.

3. Choose wells of TSR8 standard dilutions and of no target control.

4. In the [Analysis] pull down menu, select Amplification plot. On FAM window, change Base line range from 3 to 13 (default range from 3 to 15). Select Recalculate.

5. Deselect FAM and choose ROX. Repeat step 4.

(Option) If desired, print the amplification plots of TSR8 serial dilutions, No Target Control, and experimental samples in FAM and ROX window. At this step, the factors described in step 9 can be visually examined.

6. In the Analysis pull down menu, click Standard Curve. Examine overall quality of the standard curve: slope -4.0 to approx -4.5. R2 >0.98. Print if necessary.

7. From [File] menu, export the results of the experiment (.results) to a Zip drive, designated file or networked computer. Open the file by Microsoft Excel®.

8. Examine the data of Standard TSR8 dilutions and No Target Controls.

a. Examine the amplification of the internal control in the ROX window. The threshold cycle (Ct)-ROX of all the standard reactions including NTC should be within 0 to 2 cycles of each other at approx 17-19.

b. Examine the FAM window amplification (Ct-FAM) of the NTC.

10. Assess telomerase activity in the experimental samples.

a. If no amplification is observed in the FAM window of the NTC, all experimental samples with threshold cycle lower than 40 cycles are assessed as telomerase-positive.

b. If one or all of the NTC reactions shows Ct-FAM lower than 40 cycles, only samples with Ct-FAM that is at least 4 cycles fewer than that of the NTC are assessed as telomerase-positive.

11. Examination of the PCR inhibition in the samples.

a. If the Ct(ROX) of a sample is within two cycles of average Ct(ROX) obtained for TSR8 standards, the sample contains no or little PCR inhibitor(s). The data calculated automatically by 7700 can be interpreted as telomerase activity in the sample.

b. If the Ct(ROX) is more than two cycles larger than average Ct(ROX), it indi cates the presence of PCR inhibition. The telomerase activity in this sample may be underestimated. Dilute the sample extract and repeat the TRAP assay.

3.3. Example of Data Acquisition and Analysis With ABI 7700

The following results demonstrate the example of data analysis where the Texas Red-labeled internal control (registered in the ROX window) is utilized for detection of PCR inhibition with experimental samples (as described in Subheading 3.2.4.).

1. Using the experimental procedures described here, a TRAP assay with ABI PRISM 7700 was performed for analysis of five lung cancer specimens. The Ct values in FAM and ROX windows are summarized in Table 1.

2. The standard curve and its equation were determined from the data obtained for serially diluted TSR8 control target (Fig. 2). x-axis: log[TPG], y-axis: Ct(FAM). It shows a good correlation between the threshold cycle (Ct) and the TSR8 target concentration over 2.5 logs (0.64-400 TPG). Figure 3A,B are amplification plots in FAM and ROX window of the TSR8 controls utilized for generating the standard curve. The amplification plot of the Internal control (Fig. 3B) shows little variation in Ct among all TSR8 dilutions (average Ct = 16.92, stdev = 0.36). Uniform Ct for the internal control amplification reflects equal amount of target (T42) molecule in each reaction and no PCR inhibition in these reactions.

3. Based on the equation of the standard curve:

Y = -4.195 x + 33.256 (where Y is Ct[FAM], x is log[TPG]) Telomerase activities in samples 1-5 are computed automatically by the software of Sequence Detection System using the Ct(FAM) of each reaction (Table 1, column 7). Each unit of TPG (Total Products Generated) corresponds to the number of TS primers (0.001 amol or 600 molecules) extended with at least three telomeric repeats by telomerase in an experimental sample extract in a 30 min incubation at 30°C. According to the computation, all samples show low level or no telomerase activity.

4. Examine the Ct(ROX) (amplification of internal control) of the experimental samples (Table 1, column 5 and Fig. 3C). The average Ct(ROX) of TSR8 standard is 16.92 with a standard deviation of 0.36. In contrast, the Ct(ROX) of the samples 1-5 were 18.31, 28.63, 17.41, 39.99, and 21.52, respectively (Table 1). The amplification plot of the experimental samples (Fig. 3C) clearly demonstrates the difference of the Ct(ROX) between the samples and the TSR8 standard. This result indicates presence of strong PCR inhibition in sample 4 (Ct is 39.99) and some inhibition in samples 2 and 5 and suggests that telomerase-negative results obtained for these samples may possibly be false-negative owing to a PCR inhibition. On the other hand, samples 1 and 3 are telomerase-negative because no PCR inhibition was detected in the assay as judged by Ct(ROX).

5. Samples 2, 4, and 5 were diluted to 10-fold and were reanalyzed (see the data shown in last three rows in Table 1). A positive signal in FAM window was detected (Table 1, column 4). The Ct(ROX) of the diluted samples is similar to

166 Uehara Table 1

Analysis of Lung Cancer Specimens by PRISM® 7700

Quantity) log Ct Ct log (corrected for

Quantity) log Ct Ct log (corrected for

Standard

0.4 amol

400.0

2.602

22.47

17.3

0.4

400.0

2.602

22.36

16.9

0.08

80.0

1.903

25.25

17.15

0.08

80.0

1.903

25.29

16.86

0.016

16.0

1.204

27.63

16.19

0.016

16.0

1.204

28.53

16.73

0.0032

3.2

0.505

30.94

16.65

0.0032

3.2

0.505

31.23

16.49

0.00064

0.64

-0.194

34.11

17.34

0.00064

0.64

-0.194

34.24

17.11

0

0.0

40

16.97

0

0.0

40

17.37

Samples

1

36.52

18.31

-0.78

0.17

2

36.15

28.63

-0.69

0.20

3

39.82

17.41

-1.56

0.03

4

35.64

39.99

-0.57

0.27

5

37.69

21.52

-1.06

0.09

Samples

2 (1/10 dil)

39.82

17.53

-1.56

0.03

0.27

4 (1/10 dil)

29.84

18.08

0.81

6.52

65.21

5 (1/10 dil)

36.45

17.61

-0.76

0.17

1.73

those obtained for TSR8 standard reaction suggesting little/no PCR inhibition (Table 1, column 5). The telomerase activity corrected for the dilution factors indicates presence of high activity in sample 4 and low but significant activity in sample 5 (Table 1, column 8).

6. In summary, among three samples (2, 4, and 5), which appear to be telomerase-negative in initial analysis, sample 2 is true-negative, whereas samples 4 and 5 are telomerase-positive according to the second assay.

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