Nov 15, 2024
Version 1
Optimized for Routine: Highly sensitive fluorescent Telomeric Repeat Amplification Protocol (f-TRAP) V.1
- Dr. Wilhelm Dirks1,
- Silke Fähnrich1
- 1Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH
Protocol Citation: Dr. Wilhelm Dirks, Silke Fähnrich 2024. Optimized for Routine: Highly sensitive fluorescent Telomeric Repeat Amplification Protocol (f-TRAP). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8rjov5b/v1
Manuscript citation:
Submitted to BioTechniques 16th of June 2024
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working in a robust and reliable manner.
Created: June 13, 2024
Last Modified: November 15, 2024
Protocol Integer ID: 101735
Keywords: Human Telomerase, hTERT, Immortalization, fluorescent Telomeric Repeat Amplification Protocol (f-TRAP), non-radioactive, capillary electrophoresis.
Disclaimer
Publisher, editors, reviewers and authors do not accept any legal responsibility for errors, omissions or claims, nor do they provide any warranty, express or implied, with respect to information published in Inter-Research journals.
Abstract
Here we present an optimized protocol for measuring Telomerase activity that combines
a fluorescently labeled bait primer and PCR amplification with analytical
capillary electrophoresis to achieve a detection limit of one telomerase-positive
cell per ten thousand negative cells. Cellular inhibitors of the assay are
bypassed by triplicate approaches at different protein concentrations,
providing rapid results of telomerase activity with low failure rates. In
research laboratories, the simple protocol enables the assessment of
immortality in newly generated cell lines or the unambiguous evaluation of iPSC
reprogramming. The fast ad-hoc application for single assays up to high
throughput of bulk samples should make f-TRAP a promising technique prospect in
clinical oncology.
Attachments
Image Attribution
Dirks_protocolio_Fig. 1.pdf Guidelines
Background: hTERT maintains telomere length of chromosomes in germ line, stem cells and in immortal cell lines. The telomerase ribonucleoprotein is very sensitive to RNAses and temperatures! Precautions are therefore required to prevent PCR and RNase contamination. hTERT activity of cells can be measured by the presented fTRAP assay. Cell lines lacking telomerase activity such as U2OS and cell lines with active hTERT such as HeLa should be used as negative and positive control, respectively.
Materials
- Trapeze 1x CHAPS Lysis Buffer, Merck Cat. No. S7705 (Merck KGaA, Darmstadt, Germany).
- Pierce Coomassie Plus (Thermo Scientific, Cat. No. 23238) for Bradford assay to determine the protein concentration of lysates.
- Oligo Clean & Concentrator, Zymo Research Cat. No. D4061 (Irvine, USA).
- TaKaRa Taq Hot Start Version, Takara Bio Inc. Cat. No. R007A (Shiga, Japan).
- Oligonucleotides by Merck (Darmstadt, Germany).
TS (bait)(D4)5´-AATCCGTCGAGCAGAGTT (D4 is a WellRed flourescent label)
R-oriACX 5´-CTTACCCTTACCCTTACCCTAACC
- Capillary Electrophoresis: All reagents, chemicals, hardware and software from AB Sciex Germany GmbH (Darmstadt, Germany).
Safety warnings
Cellular inhibitors of the assay are bypassed by triplicate approaches at different protein concentrations, providing rapid results of telomerase activity with low failure rates. Pierce Coomassie Plus (Thermo Scientific, Cat. No. 23238) was used in a Bradford assay to determine the protein concentration of lysates. Depending on cell lines used concentrations were generally between 1.0 and 3.5 µg/µl protein. Triplicate aliquots of 10, 5, and 1 µl with equivalents for 9x104,4.5x104 and 9x103 cells, respectively, and a heat-inactivated equivalent of 4.5x104 cells were subjected to an extension reaction in a final volume of 50 µl.
Before start
The bait and forward TS primer is labeled at the 5'-end with the fluorophore D4 [WellRED-Oligos, Eurofins, Munich, Germany] in order to achieve a high extinction coefficient, which absorbs at 670 nm. Other fluorophores can also be used for labeling, provided the capillary electrophoresis is equipped with suitable filters.
Optimized for Routine: Highly sensitive fluorescent Telomeric Repeat Amplification Protocol (f-TRAP)
Optimized for Routine: Highly sensitive fluorescent Telomeric Repeat Amplification Protocol (f-TRAP)
1h
1h
LYSATE PREPARATION
1h
Count and pellet 1x106 cells of best viability and place in a 1,5 ml reaction cap.
Wash cells 1x using PBS at room temperature.
Dissolve pellet thoroughly in 50 µl TRAPeze® 1X CHAPS lysis buffer and incubate for 30 min on wet ice.
CHAPS (3-[(3-Cholamidpropyl)dimethylammonio]-1-propane sulfonate) is anon-denaturing zwitterionic surfactant for the separation of proteins from thenon-soluble parts of the cell including membrane proteins. The use of “classical”Triton-X, Tween, SDS are less efficient, especially with tissue and spheroids,and lead to significantly increased failure rates.
Centrifuge lysate for 20 min at 12.000 g at 4 °C.
Save 45 µl supernatant in a fresh cap and add 45 µl sterile water, store at -80°C or use directly.
Pierce Coomassie Plus (Thermo Scientific, Cat. No. 23238) is used to determine the protein concentration of lysates (Bradford Assay). Depending on cell lines used concentrations were generally between 1.0 and 3.5 µg/µl protein. Triplicate aliquots of 10, 5, and 1 µl with equivalents for 9x104, 4.5x104 and 9x103 cells, respectively, as well as a heat-inactivated equivalent of 4.5x104 cells were subjected to an extension reaction in a final volume of 50 µl).
EXTENSION REACTION
Cells from different tissues can differ in the quantity and quality of inhibitors for the TRAP assay. Reliable results are usually obtained with triplicate preparations and 1:10, 1:50 and 1:100 dilutions of the lysate.
1h
An aliquot of 15 µl out of 90 µl supernatant is used as negative control by heat inactivation at 85°C for 10 min.
A triplicate reaction using 10 µl lysate (corresponds to 90,000 cells), 5 µl lysate (45,000 cells) and 1 µl lysate (9,000 cells) guarantees a reliable result.
Pipette 5 µl of 10xTRAP-buffer (200 mM Tris-HCL pH8,3; 15 mM MgCl2, 630 mM KCl2; 0,005% Tween20; 10 mM EGTA) into 0,2 ml PCR caps or cap stripes for each reaction.
Add 5 µl of 10 mM dNTP (Takara) and 5 µl of 1 µM flourescent TS primer (bait).
Add per triple set 10, 5 and 1 µl lysate and 10 µl of heat-inactivated lysate to the control. Fill up to a final 50 µl volume with sterile water.
Mix thoroughly and incubate at 30 °C for 60 min in an PCR device.
Inactivate reactions for 5 min at 94°C and keep at 4°C.
PURIFICATION OF EXTENSION PRODUCTS
20m
Add to each extension reaction 100 µl oligo binding buffer of Oligo Clean & Concentrator Kit (ZymoResearch)
Add 400 µl fresh ethanol (98%) to each extension reaction
Pipet the total 550 µl to the column and centrifuge at 15 000 g for 30 sec.
Wash once using 750 µl wash buffer, centrifuge at 15 000 g for 60 sec.
Pipett 25 µl of sterile water to the bound extension products and incubate for 1 min.
Eluate extension product by centrifugation at 15 000 g for 30 seconds.
Keep the eluates on wet ice.
PCR AMPLIFICATION
2h
Place 20 µl of eluate into 0,2 PCR caps or cap stripes.
Prepare PCR reaction mix for n + 1 sample (2,5 µl 10X Takara buffer, 1 µl dNTP, 1 µl 10µM R-oriACX-Tel primer, 0,2 µl Takara Taq polymerase (1 U), 0,3 µl sterile water.
Add 5µl PCR reaction mix to each eluate, mix thoroughly and spin down for 10 sec.
f-TRAP PCR PROGRAMM
step1: 60 sec 95°C (1 cycle),
step 2: 30 sec 94 °C, 45 sec 56 °C, 45 sec 70 °C (10 cycles),
step 3: 30 sec 90 °C, 45 sec 56 °C, 45 sec 70 °C (20 cycles),
step 4: 40 min 56 °C, ~ RT.
ANALYSIS USING CAPILLARY ELECTRPHORESIS (CE)
1h
Prepare 24 µl loading mix for n + 1 sample by placing 23,5 sample loading solution (SLS) and 0,5 µl of a 600 bp size standard (AB Sciex, Darmstadt, Germany).
Add 1 µl PCR reaction of sample triplicates to device-specific loading plates for CE.
Use device-specific running condition of CE for fragment analysis between 70 and 600 nucleotide length.
In general, capillary electrophoreses provide software that enables DNA fragment analysis in order to automatically evaluate the PCR result. Depending on the manufacturer, the analysis parameters are set via dialog boxes that can be saved so that the auto-run function can be activated with a single mouse click. The analysis settings include options for detecting telomeric fragment sizes of 6 bp lengths and for correcting common chemical artifacts (pull-up, peak saturation, spike removal).
For quick access and long-term documentation, evaluated f-TRAP electropherograms can be easily and securely stored in a database.
Protocol references
1. Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 1985;43:405–13.
2. Kim NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266:2011–5.
3. Braunschweig R, Guilleret I, Delacrétaz F, et al. Pitfalls in TRAP Assay in Routine Detection of Malignancy in Effusions. Diagn. Cytopathol. 2001;25:225–230.