Download RealTime ready Cell Lysis Kit

Fast One-Step Cell Lysis
RealTime ready Cell Lysis Kit
9 | 2010
Technical Note
Accelerated Workflow for
Two-Step RT-qPCR Analysis
1 Introduction
Reverse transcription quantitative real-time PCR (RT-qPCR)
is the most commonly used method for the quantification
of mRNA expression levels.1,2
Generally, the starting material for RT-qPCR is purified
RNA or mRNA. Since RNA does not serve as a template
for PCR, it must first be copied into cDNA using a reverse
transcriptase. The cDNA is then amplified by PCR to
detectable levels for the quantification of gene expression.
RT-qPCR can be performed either as a coupled, one-step
procedure (with RT and PCR performed in a single tube)
or as a two-step procedure (with RT and PCR in separate
tubes).3
This technical note describes an accelerated workflow
for two-step RT-qPCR procedures for gene expression
profiling of cultured cells.
For life science research only.
Not for use in diagnostic procedures.
Increase convenience: Replace multi-step
RNA purification protocols with a simple
one-step cell lysis procedure.
Conventional column-based multi-step RNA
purification methods deliver high-quality RNA,
though the preparation methods are tedious
and time-consuming. Therefore, new procedures,
which streamline and ­accelerate the RNA
preparation by fast and convenient cell lysis, have
become increasingly ­popular for gene ­expression
analysis.
Improve time-to-result: Lyse samples in just
5 minutes at room ­temperature without further
steps.
Reduce intermediate steps: ­Perform the
DNase treatment during the cDNA synthesis
incubation.
The RealTime ready Cell Lysis Kit, together with
other innovative Roche cDNA synthesis and
real-time PCR products, provides an easy stream­
lined workflow for facilitating fast repro­ducible
gene expression data (see Figures 1 and 2,
and page 6).
Benefit from broad applicability: Scale up
from 3 to 30,000 cultured cells for qPCR
applications in 96- to 1536-well formats,
with or without a­ utomation.
RNA Preparation
Convenience
cDNA Synthesis
qPCR
Accelerated
Workflow
Conventional
Workflow
30
60
90
120
150
Time-to-Result (min)
Figure 1: Time savings and increased ­c onvenience in two-step RT-qPCR analysis of cell cultures.*
Simplify your experimental workflow by ­s ignificantly reducing handling steps. The Accelerated Workflow
(single-step cell lysis, fast cDNA synthesis combined with DNase treatment, qPCR) cuts your time in half
for gene expression analysis in comparison to the ­C onventional Workflow (multi-step column-based RNA
isolation, standard cDNA synthesis, qPCR).
* Calculation of time for the preparation of 96 cell culture samples.
2
1. Remove medium from the cell culture plate (96-well format).
2. Wash cells once with ice-cold PBS (+4°C).
Setup for 96-well format (Cell Lysis Reagent):
Dilute the Protector RNase Inhibitor (0.5 µl/reaction) into the Cell Lysis Buffer (39.5 µl/reaction).
4. Add 40 µl RealTime ready Lysis Reagent to each well (96-well format).
Cell Lysis
3. Prepare the Cell Lysis Reagent.*
i
5. Incubate for 5 minutes at room temperature.
10 Min.
5 Min.
➣ Store the lysates for later RT-qPCR analysis.
➣Or, proceed to cDNA synthesis.
7. Combine the cDNA synthesis mix (18 µl/reaction) with the lysates (2 µl/reaction)
in reaction plates or tubes.
8. Perform the cDNA synthesis incubation.
i
10 Min.
25 Min.
cDNA synthesis protocol:
Primer annealing/DNase treatment:
Reverse transcription:
Denaturation:
10minutes at +29°C
10minutes at +55°C
5minutes at +85°C
➣ Store the cDNA samples for later qPCR analysis.
➣ Or, proceed to qPCR analysis.
10. Distribute the qPCR mix (15 µl/well) into the pre-plated LightCycler® 480 Multiwell Plate
(RealTime ready Human Apoptosis Panel, 96) and add cDNA samples (5 µl/well).*
10 Min.
70 Min.
qPCR
9. Prepare the qPCR mix using the LightCycler ® 480 Probes Master.*
cDNA Synthesis / DNase Treatment
6.Prepare the cDNA synthesis mix using the Transcriptor Universal cDNA Master
(including thermolabile DNase).* /**/***
11. Perform qPCR on the LightCycler® 480 Instrument (standard PCR protocol).
Figure 2: Accelerated RT-qPCR Analysis – Experimental Workflow Overview.
The resulting total times (incubation time: 100 minutes, hands-on time: 30 minutes) for the depicted RT-qPCR experiment are valid only
for that particular combination of products and protocols for processing 96 cell culture samples.
Hands-on time
Incubation time
qPCR Protocols – Faster Time to Result
RealTime ready assays are function tested for standard PCR protocols in combination with the LightCycler ® 480 Probes Master for
best performance with any target of interest. Shortening of PCR protocols can be implemented by users in accordance with particular
assay needs (fast PCR protocol: < 50 minutes). Alternatively, the RealTime ready DNA Probes Master can be used for qPCR experiments
with custom assays enabling fast PCR protocols (approximately 40 minutes).
* For detailed experimental information regarding the RealTime ready Cell Lysis Kit, Transcriptor Universal cDNA Master, LightCycler ® 480 Probes
Master, or RealTime ready Human Apoptosis Panels, 96, refer to the corresponding package inserts.
** The thermolabile DNase is a component of the scope of delivery provided with the RealTime ready Cell Lysis Kit.
***Alternatively, the Transcriptor First Strand cDNA Synthesis Kit can be used for reverse transcription; an advanced protocol is described in the
package insert of the RealTime ready Cell Lysis Kit.
3
2 Materials, Methods, and Results
Cell Cultivation, Cell Growth ­Monitoring, and Compound ­A dministration
The HepG2 cell line, derived from human liver
carcinoma, was used for the following research
experimental setup. Prior to plating, cell number
and cell viability was measured using the Cedex XS
image-based automated cell culture analyzer.
HepG2 cells were seeded into E-Plates 96 at a
density of 2.5 x 104 cells/well.
Cells were plated in 200 µl MEM, ­supplemented
with 10% fetal calf serum (FCS), 1% non-essential
amino acids (NEAA), L-glutamine (2 nM),
penicillin (100 U/ml), and streptomycin (0.1 mg/ml),
and cultured at +37°C in a 5% CO2-humidified
atmosphere.
Cell growth behavior was continuously monitored
using the xCELLigence RTCA MP Instrument.
Background impedance was measured in 100 μl cell
culture medium per well. After plating, impedance
was recorded at 15-minute intervals.
After 24 hours, the HepG2 cells were treated
with different concentrations of doxorubicin
(200 nM, 2 µM, 20 µM, and 200 µM), a drug used
in chemotherapy. After compound administration,
impedance was recorded in 2-minute intervals
for 1 hour followed by 5-minute intervals for
2 hours, and 15-minute intervals for the remaining
time (see Figure 3). Cell Index (CI) values
were normalized to the time point of compound
administration (referred to as normalized CI).
Two E-Plates 96 were used in parallel for the
treatment approach: one E-Plate was continuously
monitored for 96 hours; the second experiment
was stopped 8 hours post-treatment for cell lysis
and gene expression profiling.
C ompound
Administration
Control (PBS)
200 nM Doxorubicin
2 µM Doxorubicin
20 µM Doxorubicin
200 µM Doxorubicin
Figure 3: The effect of doxorubicin on the proliferation and viability of HepG2 cells.
Higher compound concentrations (20 µM and 200 µM) introduced a faster decline in CI values and therefore cell death.
At a concentration of 2 µM of doxorubicin, the CI values first exceed the level of the control (PBS) curve and then
decrease faster than the curve representing the doxorubicin concentration of 20 µM. At a concentration of 200 nM,
the CI values again exceed the level of the control curve but stabilize approximately 24 hours post-treatment at a
constant level, indicating cell arrest.
For further details regarding the Cedex XS and the xCELLigence RTCA Instruments, visit our homepages:
 www.roche-applied-science.com/sis/innovatis/index.jsp
 www.xcelligence.roche.com
4
Cell Lysis, cDNA Synthesis, and DNase Treatment
After the completion of the experiment, the
untreated (PBS control cells) and treated HepG2
cells were washed once within the E-Plates
(100 µl ice-cold PBS). Only treatment groups with
a measured significant change in the proliferation
rate (2 µM, 20 µM, and 200 µM) were profiled in
the following RT-qPCR experiments regarding
expression of key genes of the cellular apoptotic
pathway. All samples were run in technical
triplicates through cDNA synthesis and qPCR
analysis.
For fast RNA allocation, time-consuming and
cumbersome conventional RNA preparation methods
were bypassed by using the RealTime Cell Lysis Kit
(according to the package insert: 40 µl total lysis
volume/reaction, 96-well format; see Figure 2).
This quick single-step cell lysis requires no
intervention during its short incubation time.
The resulting lysate was directly reverse transcribed
for fast cDNA synthesis (2 µl of the total lysate/
cDNA reaction) using Transcriptor Universal
cDNA Master according to the package insert
(20 µl total cDNA reaction volume, see Figure 2).
Spotlight 1: Assay Design and DNase Treatment
Amplicons from cDNA targets should be carefully
selected to prevent co-amplification of genomic
DNA (gDNA) or pseudogenes which may
contribute misleadingly to higher gene expression
values. Adequate strategies for primer design,
such as intron-exon spanning assays, should be
considered during the preparation phase of gene
expression studies.3,4
If a proper primer design cannot be performed
due to sequence-specific restrictions (e.g., oneexon genes) or the presence of one or more
processed pseudogenes of a particular target
sequence, a DNase treatment during the cDNA
synthesis step is highly recommended for the
elimination of contaminating DNA. Due to the fact
that intron-exon boundary information has not
yet been published for all genes nor all putative
pseudogenes are known, DNase treatment
has been addressed as a crucial prerequisite for
accurate RT-qPCR results. 5,6
Optionally, DNase treatment can be integrated into
the cDNA synthesis protocol (see Spotlight 1 below).
This combination of cDNA synthesis and DNase
treatment streamlines the entire experimental
workflow and reduces the overall number of single
steps, thus increasing convenience together
with minimizing error sources and sample loss
(total reaction time 25 minutes, see Figure 2).
An additional benefit provided by the RealTime
ready Cell Lysis Kit is the usage of a thermolabile
DNase that eliminates the need for an extra heating
step required by conventional approaches using
thermostable enzymes.
Overall, the use of the RealTime ready Cell Lysis Kit
accelerates the entire experimental setup of
gene expression studies by significantly reducing
handling steps and incubation times, and
eliminating the need for user intervention (e.g.,
shaking of samples, heating steps). Therefore,
this Accelerated Workflow can be easily integrated
into any automated medium- to high-throughput
workflow.
Universal ProbeLibrary (UPL) and RealTime
ready assays provide best-practice service in
primer design by considering state-of-the-art
primer design strategies, pseudogene searches,
and other in silico methods in amplicon target
sequences. For further details regarding UPL
probe design, consult our online assay design
software ( www.universalprobelibrary.com ).
All RealTime ready assays are also function tested
by qPCR experiments using the LightCycler® 480
Instrument (see Spotlight 2).
5
RealTime ready Cell Lysis Kit
Fast Gene Expression Analysis Directly from Cells
0.50 0.45 Fluorescence 465/510
0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 5
10
15
20
25
30
(Cq)
Cycles
Number of Cells
18 S
= 0.9982
ACTB = 0.9707
GAPDH = 0.9859
IP08 = 0.9961
R2 R2
R2 RPLPO = 0.9915
SDH = 0.9924
YWHAZ = 0.9882
Column-Based RNA Isolation (Cq)
R2
R2 R2 R2
RealTime ready Cell Lysis Kit (Cq)
6
35
40
45
Figure 4: Sensitivity of real-time PCR results using
the RealTime ready Cell Lysis Kit for gene expression
analysis.
Lysates generated by the RealTime ready Cell Lysis Kit
can be adapted conveniently to any throughput needs,
as shown for the 1536-well format (LightCycler ® 1536
Instrument).
Experimental setup: HeLa cells were prepared with the
RealTime ready Cell Lysis Kit and processed by reverse
transcription (Transcriptor First Strand cDNA Synthesis
Kit) and qPCR (LightCycler ® 1536 Instrument, 1 µl PCR
reaction, RealTime ready DNA Probes Master (fast PCR
protocol), five concentration steps (0.002 – 20 cell
equivalents/well), and negative controls, 96 replicates
each).
Figure 5: Linearity of real-time PCR results using
the RealTime ready Cell Lysis Kit for gene expression
analysis.
qPCR data demonstrate high performance results over
a broad range of cell equivalents for seven different
reference genes with different levels of gene expression.
Experimental setup: 30 – 30,000 MCF-7 cell equivalents/
well were used for the RNA preparation. cDNA synthesis
was done using the Transcriptor First Strand cDNA
Synthesis Kit followed by real-time PCR amplification
performed on the LightCycler ® 480 Instrument (96-well
format, 20 µl PCR reaction, RealTime ready DNA Probes
Master (fast PCR protocol), and RealTime ready Human
Reference Gene Panel).
Figure 6: Real-time PCR results using the
Accelerated Workflow with the RealTime ready
Cell Lysis Kit, and the Conventional Workflow
using a column-based RNA isolation method
(see Figure 1).
Real-time PCR findings show comparable results for
both RNA isolation techniques, resulting in high
correlation of quantification cycles (Cqs) for the
19 different reference genes tested.
Experimental setup: 30,000 HeLa cell equivalents/well
were used for the RNA preparation. RNA was reverse
transcribed using the Transcriptor First Strand cDNA
Synthesis Kit. Real-time PCR amplification was done
using the LightCycler ® 480 Instrument (96-well format,
20 µl reaction, LightCycler® 480 Probes Master, and
RealTime ready Human Reference Gene Panel).
qPCR and Data Analysis
qPCR was performed on the LightCycler® 480
Real-Time PCR Instrument (96-well) using the
RealTime ready Human Apoptosis Panel, 96 in
combination with the LightCycler® 480 Probes
Master (96-well format: 5 µl cDNA, 15 µl PCR mix,
standard PCR protocol).
The RealTime ready Human Apoptosis Panel, 96 is
part of the family of ready-to-use Focus Panels
which provide pre-plated target gene sequences for
the gene expression profiling of dedicated focus
areas (see Spotlight 2 below).
Spotlight 2: RealTime ready Focus Panels
RealTime ready Focus Panels are designed for
expression profiling of genes from a variety of
pathways, functional groups, or gene superfamilies.
Ready-to-use, function tested qPCR assays are
conveniently pre-plated and dried-down on
LightCycler ® 480 Multiwell Plates 96 or 384. The
only components that need to be added are
sample cDNA and LightCycler® 480 Probes Master
reaction mix. Each panel contains five reversetranscription (RT) controls for RNA quality:
three RT-positive controls to check the quality of
the reverse transcription step, and two RT-minus
controls to check for residual genomic DNA.
Basic Relative Quantification Analysis (∆Cq Method:
target gene-to-reference genes ratio) for each
LightCycler® 480 Multiwell Plate was performed
with the LightCycler® 480 Software (version 1.5,
Figure 7 B and C).
The following normalization of the ∆Cq values for
the different treated cell samples against the Study
Calibrator (control samples, PBS) was done by
using the LightCycler® 480 Multiple Plate Analysis
Software (∆∆Cq Method).
Seven reference gene assays serve as PCR
controls and also enable the user to perform
relative quantification of the target genes.
Each assay on the panel is function tested in
real-time PCR on the LightCycler ® 480 Instrument
to meet strict criteria:
Similar PCR efficiency of approximately 2.0.
Standard curve R2 value between 0.99 and 1.00.
Linear dynamic range of at least 3 logs.
High amplification specificity by eliminating all side products during gel analysis.
Function tested RealTime ready assays are also
available as single assays of your choice, or preplated on LightCycler ® 480 Multiwell Plates, 96
and 384. Visit our online portal for detailed
information www.realtimeready.roche.com
Nomenclature Information
The nomenclature used in this technical note is in accordance with the proposed terms of the MIQE
guidelines.6 The Cq (quantification cycle) value is equivalent to the Cp (crossing point) value.
7
Figure 7: RT-qPCR data, analysis, and results.
Sample RT-qPCR results depicted for one target gene
(BAX) in combination with seven reference genes for one
dedicated treatment group (200 µM doxorubicin).
A. Plate layout – RealTime ready Human Apoptosis Panel, 96:
The BAX gene (red) and the seven reference genes
(green) are marked by colored arrows.
B. qPCR data (LightCycler ® 480 Software, Basic Relative
Quantification Analysis): Profile of amplification curves for
the BAX gene (marked by a red arrow) together with the
seven reference genes.
C. ∆Cq results (LightCycler® 480 Software, Basic Relative
Quantification Analysis): Bar chart results of target/reference
genes (mean of: ACTB, ß2M, GAPDH, HPRT1, RPL13A, 18S,
YWHAZ) ratios. The ratio result for the BAX gene against the
seven reference genes is marked by a blue arrow.
A
B
C
Figure 8: Profile changes in gene expression due to
compound administration.
Less than 9% of all analyzed 84 apoptotic target genes
in each treatment group (2 µg: 3.5%, 20 µg: 6.0%,
200 µg: 8.3%) showed significant up-regulation
(> 3-fold change) of their gene expression level due to
compound administration. However, 10 – 29% (2 µg: 9.5%,
20 µg: 23.8%, 200 µg: 28.5%) of all genes downregulated their gene activity in response to the treatment.
8
3 Conclusion
The overview of the experimental workflow and
RT-qPCR results presented in this Technical Note
demonstrates that the RealTime ready Cell Lysis Kit
represents a beneficial building block in the
pathway of gene expression analysis by providing
significant time savings combined with increased
convenience for low- to medium-throughput,
real-time PCR applications. Together with the
Transcriptor Universal cDNA Master, with its
convenient two-component kit concept, and
function tested RealTime ready assays, even
complex gene expression studies can be made easier
and more effective, with tremendous savings
in time and resources. By supporting high-quality
results in target profiling, these state-of-the-art
products are an attractive solution for meeting
diverse research challenges, whether one is studying
a few cells, or thousands of cells.
References
1Wong ML. and Medrano F. "Real-time PCR for mRNA quantification." (2005).
Biotechniques 39 (1): 75-85.
2 Bustin SA. “Quantification of mRNA using real-time reverse transcription PCR (RT-PCR):
trends and problems.” (2002). J of Mol Endocrinol. 29: 23-39.
3PCR Applications Manual, 3rd edition. (2006). Roche Diagnostics GmbH.
4 LightCycler ® Real-Time PCR Systems – Application Manual. (2009). Roche Diagnostics GmbH.
5Vandesompele J. et al. “Elimination of Primer-Dimer Artifacts and Genomic Coamplification
Using a Two-Step SYBR Green I Real-Time RT-PCR.” (2002). Biochem. 303: 95-98.
6Bustin SA. et al. “The MIQE Guidelines: Minimum Information for Publication of Quantitative ­
Real-Time PCR Experiments.” (2009). Clin Chem. 55: 611-622.
9
Ordering Information
Product
Catalog Number
Pack Size
RealTime ready Cell Lysis Kit
06 366 821 001
05 943 523 001
50 x 40 µl reactions
500 x 40 µl reactions
Transcriptor Universal cDNA Master
05 893 151 001
100 x 20 µl reactions
Transcriptor First Strand cDNA Synthesis Kit
04 379 012 001
04 896 866 001
04 897 030 001
50 x 20 μl reactions
100 x 20 μl reactions
200 x 20 μl reactions
LightCycler® 480 Probes Master 2 
04 707 494 001
04 887 301 001
500 x 20 μl reactions
5000 x 20 μl reactions
LightCycler® 480 SYBR Green I Master    
04 707 516 001
04 887 352 001
500 x 20 μl reactions
5000 x 20 μl reactions
RealTime ready DNA Probes Master  
05 502 381 001
1,250 x 20 μl reactions
RealTime ready assays  
For detailed information, visit www.realtimeready.roche.com
Universal ProbeLibrary assays 
For detailed information, visit www.universalprobelibrary.com
xCELLigence System 
For detailed information, visit www.xcelligence.roche.com
LightCycler® Real-Time PCR Systems    
For detailed information, visit www.lightcycler480.com
www.lightcycler1536.com
Learn more about Roche’s products for gene expression analysis at:
www.gene-expression.roche.com
10
 A license to perform the patented 5’ Nuclease Process for research is
obtained by the purchase of (i) both Licensed Probe and Authorized 5’
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Applied Biosystems. This product contains Licensed Probe. Its purchase price
includes a limited, non-transferable immunity from suit under U.S. Patents Nos.
6,214,979 (exp. 8/6/2010) and 5,804,375 (claims 1-12 only, (exp. 9/8/2015) and
corresponding patent claims outside the United States. The purchase of this
product includes a limited, non-transferable immunity from suit under the
foregoing patent claims for using only this amount of product for the
purchaser’s own internal research. The right to use this product in the 5’
Nuclease Process under the applicable claims of US Patents Nos. 5,210,015
(exp. 8/6/2010) and corresponding patent claims outside the United States, can
be obtained through purchase of an Authorized 5’ Nuclease Core Kit. Except
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patents require a separate license from Roche. Further information on
purchasing licenses may be obtained from the Director of Licensing, Applied
Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
 A license to perform the patented 5’ Nuclease Process for research is
obtained by the purchase of (i) both Authorized 5’ Nuclease Core Kit and
Licensed Probe, (ii) a Licensed 5’ Nuclease Kit, or (iii) license rights from
Applied Biosystems. This product is an Authorized 5’ Nuclease Core Kit. Use of
this product is covered by one or more of the following US patents and
corresponding patent claims outside the US: 5,789,224 (exp. 4/8/2014),
5,618,711 (exp. 4/8/2014), 6,127,155 (exp. 10/3/2017), 5,677,152 (exp. 7/19/2017),
5,773,258 (exp. 7/11/2016), 5,407,800 (exp. 4/11/2011), 5,322,770 (exp.
5/10/2011), 5,310,652 (exp. 5/10/2011), 5,210,015 (exp. 8/6/2010) and claims
outside the US corresponding to US Patent No. 4,889,818 (expired in US). The
purchase of this product includes a limited, non-transferable immunity from
suit under the foregoing patent claims for using only this amount of product for
the purchaser’s own internal research. Separate purchase of a Licensed Probe
would convey rights under the applicable claims of US Patents Nos. 5,538,848
(exp. 11/16/2014), 5,723,591 (exp. 11/16/2014), 5,876,930 (exp. 11/16/2014),
6,030,787 (exp. 11/16/2014), 6,258,569 (exp. 11/16/2014), 5,804,375 (claims
1-12 only (exp. 9/8/2015)), and 6,214,979 (exp. 8/6/2010), and corresponding
claims outside the United States. No right under any other patent claim and no
right to perform commercial services of any kind, including without limitation
reporting the results of purchaser’s activities for a fee or other commercial
consideration, is conveyed expressly, by implication, or by estoppel. This
product is for research use only. Diagnostic uses under Roche patents require a
separate license from Roche. Further information on purchasing licenses may
be obtained from the Director of Licensing, Applied Biosystems, 850 Lincoln
Centre Drive, Foster City, California 94404, USA.
 Use of this product is covered by one or more of the following US patents
and corresponding patent claims outside the US: 5,789,224 (exp. 4/8/2014),
5,618,711 (exp. 4/8/2014), 6,127,155 (exp. 10/3/2017), 5,677,152 (exp. 7/19/2017),
5,773,258 (exp. 7/11/2016), 5,407,800 (exp. 4/11/2011), 5,322,770 (exp.
5/10/2011), 5,310,652 (exp. 5/10/2011), 5,994,056 (exp. 11/30/2016), 6,171,785
(exp. 11/30/2016), and claims outside the US corresponding to US Patent No.
4,889,818 (expired in US). The purchase of this product includes a limited, nontransferable immunity from suit under the foregoing patent claims for using
only this amount of product for the purchaser’s own internal research. No right
under any other patent claim (such as apparatus or system claims in US Patent
No. 6,814,934 (exp. 5/2/2011) and no right to perform commercial services of
any kind, including without limitation reporting the results of purchaser’s
activities for a fee or other commercial consideration, is conveyed expressly, by
implication, or by estoppel. This product is for research use only. Diagnostic
uses under Roche patents require a separate license from Roche. Further
information on purchasing licenses may be obtained by contacting the Director
of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City,
California 94404, USA.
 This LightCycler ® 480 Real-Time PCR System is a real-time thermal cycler
licensed for research use only under U.S. Patent No. 6,814,934 (exp. 5/2/2011)
and corresponding claims in its non-U.S. counterparts, and under one or more
of U.S. Patents Nos. 5,475,610 (exp. 12/12/2013), 5,602,756 (exp. 3/14/2011),
6,703,236 (exp. 11/29/2010), 7,238,517 (exp. 11/29/2010), 7,504,241 (exp.
11/29/2010), 7,537,377 (exp. 2/9/2020) or corresponding claims in their non-U.S.
counterparts, owned by Applied Biosystems, LLC. No rights are conveyed
expressly, by implication or estoppels to any other patent claims.
The LightCycler ® 480 Real-Time PCR System is not intended for in vitro
diagnostic use in the U.S. For further information on purchasing additional
rights, contact the Director of Licensing at Applied Biosystems, 850 Lincoln
Centre Drive, Foster City, California, 94404, USA. No right is conveyed expressly,
by implication, or by estoppels under any other patent claim, such as claims to
apparatus, reagents, kits, or methods such as 5’ nuclease methods. License
rights to practice PCR methods under the foregoing Roche patents for research
and other non-in vitro diagnostic applications may be purchased from Applied
Biosystems or may be obtained by purchasing licensed reagents from Roche,
Applied Biosystems, or other Authorized third party. For information on
purchasing licenses for research and other non-in vitro diagnostic applications,
contact the Director of Licensing at Applied Biosystems, 850 Lincoln Centre
Drive, Foster City, California 94404, USA. A license to practice PCR methods
with real-time detection under patents of F. Hoffmann-La Roche Ltd, and Roche
Molecular Systems (“Roche”) for in vitro diagnostic applications may be
purchased from Roche or may be obtained by purchasing in vitro diagnostic
reagents from Roche or any other Authorized third party. The LightCycler ® 480
Real-Time PCR System is not intended for in vitro diagnostic use in the U.S.
 This LightCycler ® 1536 Real-Time PCR System is a real-time thermal cycler
licensed for research use only under U.S. Patent No. 6,814,934 (exp. 5/2/2011)
and corresponding claims in its non-U.S. counterparts, and under one or more
of U.S. Patents Nos. 5,475,610 (exp. 12/12/2013), 5,602,756 (exp. 3/14/2011),
6,703,236 (exp. 11/29/2010), 7,238,517 (exp. 11/29/2010), 7,504,241 (exp.
11/29/2010), 7,537,377 (exp. 2/9/2020) or corresponding claims in their non-U.S.
counterparts, owned by Applied Biosystems, LLC. No rights are conveyed
expressly, by implication or estoppels to any other patent claims. The
LightCycler® 1536 Real-Time PCR System is not intended for in vitro diagnostic
use in the U.S. For further information on purchasing additional rights, contact
the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive,
Foster City, California, 94404, USA. No right is conveyed expressly, by
implication, or by estoppels under any other patent claim, such as claims to
apparatus, reagents, kits, or methods such as 5’ nuclease methods. License
rights to practice PCR methods under the foregoing Roche patents for research
and other non-in vitro diagnostic applications may be purchased from Applied
Biosystems or may be obtained by purchasing licensed reagents from Roche,
Applied Biosystems, or other Authorized third party. For information on
purchasing licenses for research and other non-in vitro diagnostic applications,
contact the Director of Licensing at Applied Biosystems, 850 Lincoln Centre
Drive, Foster City, California 94404, USA. A license to practice PCR methods
with real-time detection under patents of F. Hoffmann-La Roche Ltd, and Roche
Molecular Systems (“Roche”) for in vitro diagnostic applications may be
purchased from Roche or may be obtained by purchasing in vitro diagnostic
reagents from Roche or any other Authorized third party. The LightCycler ® 1536
Real-Time PCR System is not intended for in vitro diagnostic use in the U.S.
 This product is covered in-part by US 5,871,908 (exp. 12/16/2014) or any
foreign equivalents, co-exclusively licensed from Evotec OAI AG. The purchase
price includes a license to practice the methods covered by US 5,871,908 (exp.
12/16/2014) by using the product. Purchase of this product, however, does not
convey to the purchaser a license or right to (i) commercially make, have made
or sell reagents and/or kits, or (ii) buy or use reagents and/or kits provided by a
third party used in conjunction with the product or any other thermocycler to
practice the methods covered by US 5,871,908 (exp. 12/16/2014) or any foreign
equivalents.
 Under license from Gilead Sciences Inc.
 Parts of the Software used for the LightCycler ® 480 System are licensed from
Idaho Technology Inc., Salt Lake City, UT, USA. This product is covered by one
or more of U.S. 6,197,520 (exp. 8/13/2019), 6,303,305 (exp. 3/30/2019), 6,387,621
(exp. 9/8/2019), 6,503,720 (exp. 2/20/2021), 6,730,501 (exp. 3/13/2022) and
corresponding claims in their non-U.S. counterparts, owned by Roche
Diagnostics GmbH and/or licensed from Idaho Technology, Inc.
 ProbeLibrary is covered by US and other patent applications owned by
Exiqon A/S. Locked Nucleic Acids (LNA) are covered by U.S. Patents No US
6,794,499 (exp. 9/11/2018), US 6,670,461 (exp. 9/110/2018), US 6,268,490 (exp.
3/9/2018) & US 6,770,748 (exp. 3/9/2018) and other patents and patent
applications owned by Exiqon A/S and Prof. Takeshi Imanishi. The quencher
used in the probes is covered by patent applications owned by Exiqon A/S.
 The RTCA Analyzer in combination with either the RTCA SP Station or the
RTCA MP Station and an RTCA Software Package 1.x, with the E-Plate 96 is a
real-time cell based assay system covered by US patent No. 7,192,752 (exp.
11/10/2023), No. 7,470,533 (exp. 11/10/2023), No. 7,560,269 (exp. 10/24/2025).  SYBR Green I is manufactured by Molecular Probes, Inc., and is provided
under license from Molecular Probes, Inc., for direct research use for PCR,
where the dye is present during the PCR.
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Trademarks
Published by: CEDEX, LIGHTCYCLER, REALTIME READY, and XCELLIGENCE are trademarks of Roche.
E-PLATE and ACEA BIOSCIENCES are registered trademarks of ACEA Biosciences, Inc.
in the U.S.ProbeLibrary is a registered trademark of Exiqon A/S, Vedbaek, Denmark.
SYBR is a trademark of Molecular Probes, Inc.
Other brands or product names are trademarks of their respective holders.
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82372 Penzberg
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For life science research only.
Not for use in diagnostic procedures.
© 2010 Roche Diagnostics GmbH
All rights reserved.