Technical Note

Accelerated Workflow for Two-Step RT-qPCR Analysis

1 Introduction

For life science research only. Not for use in diagnostic procedures.

Reverse transcription quantitative real-time PCR (RT-qPCR) is the most commonly used method for the quantifi cation 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 fi rst be copied into cDNA using a reverse transcriptase. The cDNA is then amplifi ed by PCR to detectable levels for the quantifi cation 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 profi ling of cultured cells.

Fast One-Step Cell Lysis RealTime ready Cell Lysis Kit

9 | 2010

2

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. 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 repro ducible gene expression data (see Figures 1 and 2, and page 6).

Increase convenience:�� Replace multi-step RNA purification protocols with a simple one-step cell lysis procedure.

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.

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 automation.

Figure 1: Time savings and increased convenience in two-step RT-qPCR analysis of cell cultures.*Simplify your experimental workflow by significantly 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 Conventional Workflow (multi-step column-based RNA isolation, standard cDNA synthesis, qPCR).* Calculation of time for the preparation of 96 cell culture samples.

Convenience

30

ConventionalWorkflow

AcceleratedWorkflow

60 90 120 150Time-to-Result (min)

RNA Preparation

cDNA Synthesis

qPCR

3

* 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.

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.

qPCR Protocols – Faster Time to ResultRealTime 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).

Hands-on time Incubation time

qP

CR

Cell Lysis

cDN

A S

ynth

esis / DN

ase Treatmen

t

5 Min.

70 Min.

25 Min.

10 Min.

10 Min.

10 Min.

1. Remove medium from the cell culture plate (96-well format).

2. Wash cells once with ice-cold PBS (+4°C).

3. Prepare the Cell Lysis Reagent.*

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).

5. Incubate for 5 minutes at room temperature.

➣ Store the lysates for later RT-qPCR analysis. ➣ Or, proceed to cDNA synthesis.

6. Prepare the cDNA synthesis mix using the Transcriptor Universal cDNA Master (including thermolabile DNase).*/**/***

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.

cDNA synthesis protocol: Primer annealing/DNase treatment: 10 minutes at +29°C Reverse transcription: 10 minutes at +55°C

Denaturation: 5 minutes at +85°C

➣ Store the cDNA samples for later qPCR analysis. ➣ Or, proceed to qPCR analysis.

9. Prepare the qPCR mix using the LightCycler® 480 Probes Master.*

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).*

11. Perform qPCR on the LightCycler® 480 Instrument (standard PCR protocol).

i

i

4

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.

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.

2 Materials, Methods, and Results

Cell Cultivation, Cell Growth Monitoring, and Compound Administration

Compound Administration

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

Control (PBS) 200 nM Doxorubicin 2 µM Doxorubicin 20 µM Doxorubicin 200 µM Doxorubicin

5

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 signifi cant change in the proliferation rate (2 µM, 20 µM, and 200 µM) were profi led 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).

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 workfl ow 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 Workfl ow can be easily integrated into any automated medium- to high-throughput workfl ow.

Cell Lysis, cDNA Synthesis, and DNase Treatment

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., one-exon 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

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).

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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 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).

RealTime ready Cell Lysis Kit Fast Gene Expression Analysis Directly from Cells

RealTime ready Cell Lysis Kit (Cq)

Col

umn-

Bas

ed R

NA

Isol

atio

n (C

q)

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).

R2 18 S = 0.9982

R2 ACTB = 0.9707

R2 GAPDH = 0.9859

R2 IP08 = 0.9961

R2 RPLPO = 0.9915

R2 SDH = 0.9924

R2 YWHAZ = 0.9882

Number of Cells

(Cq)

0.50

0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

Fluo

resc

ence

465

/510

Cycles

5 10 15 20 25 30 35 40 45

7

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).

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).

qPCR and Data Analysis

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.

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 reverse-transcription (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.

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 R�� 2 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 pre-plated on LightCycler® 480 Multiwell Plates, 96 and 384. Visit our online portal for detailed information www.realtimeready.roche.com

8

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.

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 down-regulated their gene activity in response to the treatment.

A

C

B

9

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 1 Wong 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: 23-39.3 PCR Applications Manual, 3rd edition. (2006). Roche Diagnostics GmbH.4 LightCycler® Real-Time PCR Systems – Application Manual. (2009). Roche Diagnostics GmbH.5 Vandesompele 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: 95-98.6 Bustin SA. et al. “The MIQE Guidelines: Minimum Information for Publication of Quantitative

Real-Time PCR Experiments.” (2009). Clin Chem. 55: 611-622.

3 Conclusion

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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 Kit04 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 2 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

Ordering Information

11

A license to perform the patented 5’ Nuclease Process for research is obtained by the purchase of (i) both Licensed Probe and Authorized 5’ Nuclease Core Kit, (ii) a Licensed 5’ Nuclease Kit, or (iii) license rights from 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 under separate license rights available from Applied Biosystems, no right under any other patent claim, or to perform commercial services of any kind, including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, or to sublicense, repackage with other products, or resell in any form, 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.2 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, non-transferable 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.

Published by:

Roche Diagnostics GmbH Roche Applied ScienceWerk Penzberg 82372 PenzbergGermany © 2010 Roche Diagnostics GmbH All rights reserved.

Trademarks

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.

For life science research only. Not for use in diagnostic procedures.06

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