For Research Use Only Not for Diagnostic Use IMPORTANT NOTES BEFORE USING THE
Telomerase is a ribonucleoprotein, and as such, great care should be taken
to not introduce ribonucleases (RNases) into the reaction. Hands and dust
particles are the most common sources of RNase. Therefore the reaction
should be set up in a biological safety hood or PCR workstation, disposable
TeloExpress Quantitative gloves should be worn and disposable plasticware should be used.
Telomerase Detection Kit Aliquot TeloExpress Lysis Buffer
100 X 25 μl Reactions The biggest source of problems in the TeloExpress assay come from lysis
buffer instability and contaminations inadvertently introduced into the
TeloExpress Lysis buffer. Before you begin, aliquot the TeloExpress Lysis
INTRODUCTION buffer into several tubes so that you never have to reuse an opened tube.
TeloExpress Lysis Buffer should be stored at -20ºC, but is stable at 4ºC for
The ends of linear human chromosomes terminate with telomers that consist up to 2 months.
of a tandem repeat of the sequence TTAGGG. The telomerase enzyme is a
ribonucleoprotein that synthesizes these telomeric DNA repeat sequences.
Telomerase activity is low in healthy cells and increased in a variety of Reagents and Equipment to be Supplied by the
malignancies and in immortalized cells. User
The TeloExpress kit is an optimized and validated real-time PCR
Kit for the quantification of human telomerase activity that can be utilized in
most real-time PCR platforms. In this telomeric repeat amplification protocol • Disposible gloves
(TRAP), the telomerase activity in cell extracts adds the telomeric (TTAGGG)
repeat sequences to the 3’-end of an oligonucleotide substrate. The number • Pipettors and sterile disposable tips
of repeat sequences added by the enzyme is quantified using real-time PCR
by measuring the increase in SYBR green fluorescence upon binding to • Sterile microfuge tubes (samples) and 10-15 ml tubes (PCR
DNA. Real-time PCR is conducted under conditions in which primer dimer mixture)
formation is negligible and the extent of repeat amplification is directly
proportional to telomerase activity. • Microfuge
• Optically-clear 96-well plastic PCR plates and sealing film
KIT CONTENTS • Centrifuge and plate adaptors to spin down 96-well PCR plates
Component Contents per Kit • Real-time PCR instrument calilbrated for SYBR green I dye
TeloExpress Master Mix 2 vials of 0.75 ml Including ABI Prism 7000,7700 & 7900HT, ABI 7300 & 7500 Real-
TeloExpress Telomerase Control Oligo 1 vial of 50 μl at 1 amol/μl Time PCR Systems; ABI GeneAmp 5700; Bio-Rad iCycler;
RNase-free Water 1 vial of 1 ml Stratagene Mx3000P, Mx3005P & Mx4000; Corbett Research
ROX Reference Dye 1 vial of 50 μl at 25 μM Roto-Gene; MJ Research DNA Engine Opticon, Opticon 2 &
TeloExpress Lysis Buffer 2 vials of 1 ml Chromo 4 Real-Time Detectors; and the Cepheid Smart Cycler
• DC Protein Assay Kit (BioRad)
• Spectrophotometer capable of reading samples at 750 nM and
STORAGE CONDITIONS cuvettes
The kit is shipped at 2-8ºC and with the exception of the TeloExpress Lysis
Buffer, is stable at this temperature for up to six months. For long-term
TELOEXPRESS QUANTITATIVE TELOMERASE
storage keep the kit at -20ºC. The lysis buffer should be aliquoted upon first DETECTION KIT PROTOCOL
use and stored at -20ºC. Teloexpress Lysis Buffer is stable for 2 months at
4ºC. Avoid multiple freeze-thawing of the kit.
SAFETY INFORMATION 1. Trypsinize cultured cells, inactivate the trypsin by the addition of an
equal volume of media and count the cells in a hemocytometer.
The MSDS for this kit is available online at www.expressbiotech.com. Spin down the cells (ex. 500g X 3 min), wash the pellet with PBS,
repellet the cells and resuspend the pellet in ice-cold TeloExpress
TECHNICAL ASSISTANCE 2. Resuspend the pellet in 200 μl ice-cold TeloExpress Lysis buffer
per million cells or 100 mg tissue sample. Incubate on ice 30 min.
Please refer any technical questions to email@example.com. 3. Spin the sample at 12,000g for 3 min at 4ºC and transfer the
supernatant to a fresh tube. This step helps remove cell debris
from the extract.
4. Respin the sample at 12,000g for 20 min and transfer the
supernatant to a fresh tube. Aliquot the extract into several tubes,
quick-freeze them in a dry ice/ethanol bath or liquid nitrogen and
freeze them at -80ºC until needed.
PCR Reaction Mixture for ABI7500 & Stratagene Mx3000, Mx3005P &
5. Determine the protein concentration in the extract using the DC Mx4000 Instruments
Protein Assay Kit. If the concentration of the extract is greater than
1,100 ng/μl then it will have to be diluted with TeloExpress Lysis Component Volume
Buffer before the assay is performed in order to be in the working TeloExpress Master Mix 15 μl
concentration range of the assay which is 8.8-1,100 ng/μl RNase-free Water 8.95 μl
(corresponds to 80-5,000 cells). ROX Reference Dye 0.05 μl
Total Volume per Reaction 24.0 μl
Final ROX concentration of 50 nM
Telomerase Control Oligo Standard
PCR Reaction Mixture for Instruments Not Utilizing a ROX Reference
The Telomerase Control (TC) oligo standard provided in the kit contains 1
amol/μl of oligo in TeloExpress Lysis Buffer. Prepare 50 μL of four ten-fold
dilutions of the TC oligo standard in TeloExpress lysis buffer to provide
TeloExpress Master Mix 15 μl
standards at 1e-1, 1e-2, 1e-3 and 1e-4 amol/μl. These diluted standards are a
RNase-free Water 9 μl
stable at 2-8ºC for one week.
Total Volume per Reaction 24.0 μl
The BioRad iCycler requires 50 nM fluorescein NIST-
Telomerase Heat-Inactivated Control tracable standard (sold separately, Invitrogen) in the
If desired, the telomerase extract prepared can be heat-inactivated and used
as an additional negative control in the reaction. Heat a tube containing the 4. Add 1 μl/well of TeloExpress Lysis Buffer as the no template controls, TC
telomerase extract at 95ºC (94-98ºC) for 10 min, place on ice for 2 min. and oligo standard (1, 1e-1, 1e-2, 1e-3 & 1e-4 amol/Ul) and test article
spin the sample down (12,000g X 10 sec) before use. telomerase extracts to the PCR plate. The use of triplicate samples is
recommended. The use of more than 1 μl as a sample volume will lead to
higher Ct values and higher sample-to-sample variability and is not
recommended. As in all 96-well applications, the inner wells of the plate
should be utilized for PCR first in order to avoid edge effects. Use the corner
Telomerase Assay Protocol wells of the plate last for the same reason.
5. Add 24 μl of the diluted PCR reaction mixture to the plate, spin down the
1. Spin down kit tubes before use (12,000g X 5 sec). Thaw telomerase plate (500g X 30 sec), place it in the PCR instrument and start the program.
extracts on ice (~15 min) and prepare the TC Oligo standard dilutions as The use of a multichannel pipettor is recommended for adding the PCR
described above. mixture to the plate.
2. Program your PCR instrument as follows using SYBR green detection and 6. Use your real-time PCR instrument’s software to plot threshold cycle (Ct)
choose ROX as the reference dye, if appropriate for your instrument: versus TC oligo copy number for the oligo standards to determine the amount
of repeat sequences added (amol) in your unknown telomerase extract
samples. Only use data that lie within the Ct values of the working range of
Step Temperature Time the standard curve. The resulting data (amol repeat sequence added) is a
Telomerase Reaction 25ºC 20 min quantitative measure of the telomerase activity of your extract.
Polymerase Activation 95ºC 10 min 7. Normalize the telomerase activity to one of your test conditions to quantify
35 Cycles of: the differences in the activity of your samples.
Denaturation 95ºC 30 sec
Annealing/Extension 60ºC 90 sec
Note: The telomerase reaction will begin as soon as the oligo substrate in Data Analysis and Assay Performance
the TeloExpress Master Mix is added to the extracts. As such, the PCR
instrument needs to be programmed and ready to run before the reactions
are set up. An example of the results anticipated and the transformations made is shown
below in which five serial 10 fold dilutions of the telomerase control oligo and
four five-fold dilutions of a telomerase extract were examined. The extract
3. Prepare the diluted PCR reaction mixture as follows:
was prepared as described above using telomerase positive control cells
(Cat. No. XT-106, Huh7) with each 1 μl of undiluted extract corresponding to
5,000 cells and containing 1,100 ng protein in the DC protein assay. The
PCR Reaction Mixture for ABI 7000, 7300, 7700 & 7900HT Instruments
experiment was performed on three different days using four replicates of
each test dilution and of no template control. An ABI 7000 instrument was
employed using auto Ct and auto baseline settings in the software.
TeloExpress Master Mix 15 μl
RNase-free Water 8.5 μl
ROX Reference Dye 0.5 μl
Total Volume per Reaction 24.0 μl
Final ROX concentration of 500 nM
29 Product Catalogue Number
27 TeloExpress Master Mix XT-101
TeloExpress Telomerase Control Oligo XT-102
21 TeloExpress Lysis Buffer XT-103
19 RNase-free Water XT-104
17 ROX Reference Dye XT-105
1.00E-04 1.00E-03 1.00E-02 1.00E-01 1.00E+00
Telomerase Positive Control Cells XT-106
TC Oligo (am ol)
In the figure above the mean +/- SD values of Ct vs. Log telomerase control
oligo sequence from one experiment is plotted. In three independently
performed experiments the mean +/- SD r = 0.9991 +/- 0.00110. The NTC
Ct = 32.9 +/- 0.437. TROUBLESHOOTING
The correlation between log telomerase activity in an extract and
Ct is also highly linear in the working range of the assay (80-5,000 cells =
Problem Possible Cause Solution
8.8-1,100 ng extract). In three independently performed experiments the Signals present in Reagents contaminated Take precautions to
mean +/- SD r = 0.9957 +/- 0.00390. no template controls. avoid sample contamination.
Aerosol-barrier pipette tips may
help.Use a fresh tube of
TeloExpress Lysis Buffer (XT-103).
No amplification curve appears No PCR product Recheck protocol and instrument
for TC oligo standard settings. Run samples on a gel to
R elative T elo m erase E xtract
120 see if the PCR worked.
100 No amplification of telomerase Enzyme inactivated RNase contamination or heating of
Protein extracts telomerase extracts may destroy its
A Protein content too low Recheck and possibly increase
protein content of reactions
60 B Re-extract samples Confirm your techniques using
telomerase positive cells (XT-106).
PCR products on gel, but not in Instrument settings Recheck instrument settings
20 Real-time PCR SYBR green calibration Calibrate machine for SYBR green
1 2 3
Protein 5 25 75
A 3.78 17.94 83.19
B 4.16 22.09 63.22 CONTRACT RESEARCH
C 4.29 27.44 99.93
Need a hand with the assay? We cam run your telomerase assays for you.
The transformation of the highly linear Ct data into amol telomerase repeat
sequences added increases the variation in the assay, but allows for a Contact Information:
reproducible and accurate estimation of telomerase activity. In the figure
above, the mean +/- SD amol telomerase repeat sequence product added to Express Biotech International
the oligo substrate was determined in three separate experiments (A, B, C) 503 Gateway Drive West
using four five-fold dilutions of telomerase extract. Normailzation was Thurmont, MD 21788 USA
conducted by dividing the mean amol product produced at each dilution by Tel: 301-228-2444
the mean amol product produced at the lowest dilution tested to compare the Fax: 301-560-6570
experimentally-derived data from the true normalized telomerase input of 5, www.expressbiotech.com
25 and 75 fold of the lowest dilution (set to 1). The SDs were transformed firstname.lastname@example.org
using SD = transformed mean X (CV%/100%). The experimentally
determined values in experiments A, B and C relative to the true telomerase
content (protein) are shown. The average error CV = 19.9 +/- 0.0814%.