For Research Use Only Not for Diagnostic Use

Document Sample
For Research Use Only Not for Diagnostic Use Powered By Docstoc
					For Research Use Only             Not for Diagnostic Use                                IMPORTANT NOTES BEFORE USING THE
                                                                                               TELOEXPRESS ASSAY

                                                                                   General Considerations
                                                                                   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
                    Catalog# XT-100
                 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.
                                                                                   Extract Preparation
                    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                         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
                                                                                             Lysis buffer.

                  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                    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
                                                                                   Component                                           Volume
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.
Hot-start DNA
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
Component                                           Volume
                                                                                   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
                                                                                                                                             RELATED PRODUCTS
                                                                                                       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).
                                           40                                                           C
                                                                                                                  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,                             
25 and 75 fold of the lowest dilution (set to 1). The SDs were transformed                              
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%.