QPCR Solutions. Our QPCR solutions meet your current research needs, offer flexibility for future research goals, and match your budget.

Transcription

1 QUANTITATIVE PCR QPCR Solutions Our QPCR solutions meet your current research needs, offer flexibility for future research goals, and match your budget. INSTRUMENTS SOFTWARE REAGENTS

2 QPCR SOLUTIONS > Total QPCR Solutions Stratagene provides a total solution approach to real-time quantitative PCR (QPCR), simplifying the challenges you face in sample preparation and data analysis. Whether you are new to or experienced in QPCR, your individual needs are met with our comprehensive range of products and industry-leading support. Those getting started in QPCR benefit from our on-site user group meetings and web-based training programs, premixed reagent kits, and turnkey instrument installation. More experienced QPCR users appreciate the flexibility of our powerful, user-friendly software as well as reagent kits that support user customization and demanding assay optimization. CONTENTS: Gene Expression Process Stabilized Cell Lysates for Gene Expression Analysis Highly Pure RNA, DNA-Free Pure RNA from Difficult Samples Reverse Transcription and cdna Synthesis Universal QPCR Total RNAs QPCR and QRT-PCR Kits QPCR Instrumentation & Analysis Software Ordering Information

3 RNA Stabilization SideStep Lysis and Stabilization Products RNA & mrna Purification cdna Synthesis Absolutely RNA Formalin-Fixed Paraffin-Embedded Tissue Kit Absolutely RNA Miniprep Kit Absolutely RNA Microprep Kit Absolutely RNA NanoPrep Kit SideStep mrna Enrichment Kit SideStep QPCR cdna Synthesis Kit StrataScript QPCR cdna Synthesis Kit RNA Controls Stratagene QPCR Total RNA Reference Alien RNA Spike-In Controls Amplification Brilliant Multiplex Master Mix Brilliant Probe-Based QPCR & QRT-PCR Kits Brilliant SideStep QPCR and QRT-PCR Kits Brilliant SYBR QPCR & QRT-PCR Kits FullVelocity SYBR QPCR & QRT-PCR Kits Detection Mx3005P QPCR System Mx3000P QPCR System Analysis MxPro QPCR Software Figure 1 GENE EXPRESSION PROCESS

4 2>3 STABILIZED CELL LYSATES FOR GENE EXPRESSION ANALYSIS > SIDESTEP PRODUCTS Obtain RNA and DNA Directly from Cells Without Loss SideStep lysis and stabilization products ensure accurate gene expression by eliminating sample loss and degradation. The SideStep method employs a patent-pending buffer that lyses cells and immediately stabilizes the released nucleic acids for months. The SideStep method allows you to completely avoid nucleic acid purification and proceed directly to real-time QPCR detection, mrna extraction, and cdna synthesis. This method is ideal for sirna knockdown validation, mirna detection, and mrna profiling among a host of other cell-based applications. We offer a suite of optimized kits containing the SideStep buffer technology. SideStep Lysis and Stabilization Process Our SideStep Lysis and Stabilization Buffer a lyses cells in 10 minutes at room temperature, with the actual lysis step taking only 1 minute. RNA, mirna, and DNA are released from the cells and immediately stabilized. RNA remains intact and high quality even after six months of -20 C storage in the lysate (Figure 2). Direct quantitative reverse transcription PCR (QRT-PCR) from the SideStep lysates shows equivalent yields compared to column-purified formats (Figure 3). The SideStep buffer offers the best cell lysis and is easier to use than other cell lysis methods. Our standard SideStep protocol permits lysis of up to 1 million cells in 100 µl and is very scalable. Importantly, you can use up to 30 percent of the lysate without worrying about affecting first-strand cdna synthesis efficiency. mrna Direct from Cells The SideStep mrna Enrichment Kit a couples the SideStep lysis and stabilization buffer with our Absolutely mrna Purification Kit for efficient mrna enrichment directly from cells without having to first purify total RNA. mrna is purified directly from the lysate in 10 minutes using an engineered oligo-dt magnetic bead method after completing the quick SideStep lysis and stabilization protocol. PANEL A PANEL B Figure 2 SIDESTEP CELL LYSATES STABLE UP TO SIX MONTHS We purified total RNA from the six-month old SideStep lysate and analyzed it using an Agilent bioanalyzer. Our data clearly show no degradation of RNA, indicated by sharp peaks for the 28S and 18S ribosomal bands and a RIN = 8.5. Figure 3 EQUIVALENT YIELD TO COLUMN-BASED METHODS We used one-step QRT-PCR with a GAPDH Probe to analyze mrna in a SideStep lysate (blue lines) stored for six months at -20 C versus purified RNA (red lines) stored at -80 C. Our data clearly demonstrate RNA is stable and show no shift in Ct. The reaction efficiency was 91% with an Rsq = (Mx3000P QPCR System)

5 cdna Synthesis Directly from Cells The SideStep QPCR cdna Synthesis Kit a,b employs direct synthesis of cdna from cells without having to first isolate RNA. This highly sensitive and reliable cdna synthesis method takes only 15 minutes to perform using SideStep cell lysates and allows you to generate cdna expressly for QPCR analysis. Figure 4 shows sensitivity down to single-cell equivalents in a two-step QRT-PCR format. This high sensitivity is due to our QPCR-grade and nuclease-free StrataScript Reverse Transcriptase d as well as a highly efficient cdna synthesis buffer. Additionally, the cdna synthesis reagents are formatted in a convenient master mix for speed and high reliability. SideStep Technology in QPCR and QRT-PCR Format Rapid and precise real-time analysis directly from cell lysates is now possible with our Brilliant SideStep QPCR and QRT-PCR Kits a,b,c. Our application-tested kits employ detection of RNA using both one-step and two-step formats (Table 1). Ideally, RT-PCR is performed using primers designed to span intron-exon boundaries. Genomic DNA, mirna, and RNA can all be measured directly from SideStep lysates. RNA is stable in the lysate for up to six months, which is ideal for a host of cell-based gene expression applications such as mrna profiling and sirna knockdown validation. FIGURE 4 SENSITIVE DETECTION OF RNA USING STABILIZED LYSATES The Brilliant SideStep QRT-PCR Kit was used to analyze BAX gene expression in HeLa cells. 700 cells were lysed in the SideStep Lysis and Stabilization Buffer and converted into cdna with the cdna synthesis module (StrataScript QPCR First Strand cdna Synthesis Kit). A range of cell equivalents was removed and subsequently detected in real time using the QPCR module (Brilliant SYBR Green QPCR Master Mix). SIDESTEP PRODUCTS NUCLEIC ACID APPLICATION Brilliant SideStep QRT-PCR Master Mix, 1-Step RNA Complete kit for real-time QRT-PCR analysis of RNA, using a one-step RT-PCR format with probes Brilliant SYBR Green SideStep QPCR Master Mix Genomic DNA Complete kit for real-time QPCR analysis of genomic DNA Brilliant SYBR Green SideStep QRT-PCR Master Mix, 2-Step RNA Complete kit for real-time QRT-PCR analysis of RNA, using a two-step RT-PCR format with SYBR Green SideStep Lysis and Stabilization Buffer n/a Creation of cell lysates consisting of RNA, mirna, and DNA for subsequent analysis SideStep mrna Enrichment Kit mrna Complete kit for mrna purification directly from cells SideStep QPCR cdna Synthesis Kit cdna Complete kit for cdna synthesis directly from cells (QPCR reagents not included) Table 1 SIDESTEP KITS SUPPORT A VARIETY OF QPCR APPLICATIONS

6 4>5 HIGHLY PURE RNA, DNA-FREE > ABSOLUTELY RNA PURIFICATION KITS Pure RNA from Cells and Tissues nd match y Our Absolutely RNA Purification Kits generate RNA of high quality and purity for all QPCR and QRT-PCR experiments. Highly pure, DNA-free RNA from as little as one cell or up to 40 mg of tissue is extracted through a custom-designed spin cup. DNase treatment is done on-column and ethanol precipitation is eliminated. Pure RNA with On-Column DNase Treatment Our Absolutely RNA Purification Kits isolate highly pure, DNA-free RNA from cells, fresh or frozen tissue, formalin-fixed paraffin-embedded tissue and laser micro-dissected (LMD) cells. RNA purified using this silica-based method is free of genomic DNA and other impurities that can hamper your downstream analysis. The Absolutely RNA kits employ a guanidine thiocyanate solution to lyse cells and prevent RNA degradation, and custom-designed spin cups with a silica-based fiber matrix to bind RNA and eliminate all impurities. DNase is included and treatment is performed directly on-column. The Absolutely RNA method is superior to phenol-based methods with respect to purity and quality (Figure 5). mrna Enrichment from Total RNA The Absolutely mrna purification kit isolates pure, high-quality mrna from total RNA using a fast and simple 20-minute protocol. The Absolutely mrna method utilizes precision engineered oligo-dt magnetic bead particles that offer exceptional binding capacity. The high binding capacity combined with an optimized buffer system allows more mrna to hybridize per bead, maximizing mrna yield and minimizing rrna contamination that can confound yield determination (Table 2). The Absolutely mrna kit is completely scalable for up to 1 mg of total RNA samples. KIT METHOD YIELD of mrna/ rrna ACTUAL mrna YIELD PROTOCOL LENGTH 100 µg TOTAL CONTAMINATION (%) SUBTRACTING rrna (minutes) RNA (µg)* CONTAMINATION (µg) Absolutely mrna Kit Competitor P Competitor I Competitor Q Competitor A Oligo dt magnetic particles 3.45 less than 1% 3.45 <20 Oligo dt magnetic bead % Oligo dt sepharose % Oligo dt latex % Oligo dt magnetic bead % *mrna purification was performed in duplicate with our Universal Rat Reference RNA following each manufacturer's recommended purfication protocol. Figure 5 CLEAN RNA RNA purified with Absolutely RNA Purification Kits (A) is cleaner than RNA from TRI Reagent Method (B) (Agilent bioanalyzer). Table 2 HIGH YIELD OF PURE mrna The Absolutely mrna Purification Kit delivers a high yield of the purest poly A+ RNA using one of the fastest protocols compared to other kits on the market for mrna purification. Actual mrna yield is calculated for each method based on subtracting out the rrna contamination.

7 Pure RNA from Difficult Samples Archived tissue specimens are rich sources of material for extensive analysis of mrna expression utilizing QRT-PCR. However, the chemical nature of fixatives and fixation time significantly affects the quality of RNA and DNA extracted from these tissues. Moreover, reliable recovery of RNA from small cell populations, such as LMD cells, are also particularly challenging to analyze by QRT-PCR. Formalin-Fixed Paraffin-Embedded Tissue (FFPE) The Absolutely RNA FFPE Kit delivers a reliable, easy to use, non-toxic method to isolate quality RNA from difficult paraffin-embedded tissue sections and includes a quantitative total RNA reference standard for meaningful QRT-PCR expression analysis. Using a standard proteinase K digestion protocol is ineffective at reversing the RNA modifications caused by the fixation process, resulting in poor and unreliable RNA recovery. In contrast, our method completely reverses the RNA modifications and therefore more RNA is accessible for real-time QRT-PCR analysis than with other kits. High, medium, and low abundance genes can be quantified reliably and calibrated against the QPCR-grade RNA reference (Figure 6). Laser-Microdissected Cells Laser micro-dissection (LMD) is a technique used to isolate a pure subpopulation of cells from tissue sections containing a heterogeneous mixture for detailed molecular analysis. Isolating high-quality total RNA from LMD-recovered cells can be challenging and inefficient when standard methods are used. In contrast, our Absolutely RNA Microprep and Nanoprep Kits effectively isolate pure RNA from small samples while avoiding ethanol precipitation. The Absolutely RNA nanoprep kit is the only kit qualified to isolate RNA from a single cell. It elutes pure RNA into small, 10-µl elution volumes using our specially designed spin cups (Table 3). Reagent ABSOLUTELY RNA ABSOLUTELY RNA ABSOLUTELY RNA 96 ABSOLUTELY RNA ABSOLUTELY RNA FFPE KIT MINIPREP KIT MICROPREP KIT MICROPREP KIT NANOPREP KIT Sample Size 10 5 to 10 7 cells or 5-40 Up to 5 x 10 5 cells Up to 5 x 10 5 cells 1 x 10 4 cells down to Formalin-fixed paraffin mg of tissue a single cell embedded tissues Format Silica fiber matrix Silica fiber matrix Silica fiber matrix Silica fiber matrix Silica fiber matrix spin cup spin cup spin cup 96-well format spin cup spin cup Capacity > 200 µg RNA 50 µg RNA 50 µg RNA Approx. 200 ng RNA 50 µg RNA Elution Volume 50 µl 30 µl 30 µl 10 µl 30 µl Figure 6 THE ABSOLUTELY RNA FFPE KIT EXTRACTS RNA OF SUFFICIENT YIELD AND CONCENTRATION FOR DOWNSTREAM QRT-PCR ANALYSIS Cyclophilin gene expression level was determined using RNA isolated from various formalin fixed paraffin-embedded (FFPE) human tissues using the Absolutely RNA FFPE Kit. The Stratagene QPCR Total Human Reference RNA was used to calibrate expression levels. (Mx3000P QPCR System) Table 3 CHOOSE THE ABSOLUTELY RNA KIT THAT SUITS YOUR SAMPLE TYPE AND SIZE

8 6>7 REVERSE TRANSCRIPTION AND cdna SYNTHESIS > STRATASCRIPT QPCR FIRST STRAND cdna SYNTHESIS KIT More Sensitive cdna Synthesis for Two-Step QRT-PCR Applications Following RNA purification, or cell lysate generation, one-step or two-step QRT-PCR is employed. For the analysis of multiple genes from a single sample, RNA is converted into cdna. An aliquot of cdna can then be removed and PCR-amplified in real time. Our cdna synthesis and two-step QRT-PCR kits are exclusively master mix formats for highest reproducibility and sensitivity. Importantly, all are qualified for use in real-time applications. cdna Synthesis for QPCR The StrataScript QPCR First Strand cdna Synthesis Kit b is designed for high-efficiency conversion of RNA to cdna and is fully optimized for real-time quantitative PCR applications (Figure 7). The high sensitivity obtained with this kit is due to the inclusion of our QPCR-grade StrataScript Reverse Transcriptase and a high-efficiency cdna synthesis buffer. The StrataScript QPCR first strand cdna synthesis kit is formatted as a master mix that reduces pipetting steps and enhances reproducibility in two-step QRT-PCR. It also employs a fast, 15-minute protocol for most targets. Robust Reverse Transcription Our StrataScript reverse transcriptase (RT) synthesizes a complementary DNA strand from single-stranded RNA, DNA, or an RNA:DNA hybrid. This enzyme has been genetically engineered to remove RNase H activity without affecting the desired reverse transcriptase function. As a result, the StrataScript RT generates robust cdna yields, longer transcripts than MMLV and AMV, and is the ideal enzyme for QRT-PCR (Figure 8). Two-Step QRT-PCR Kits Using StrataScript QPCR cdna Synthesis Kit Achieve sensitive and specific QRT-PCR in a flexible two-step format. These kits are composed of two modules. First, RNA is reverse-transcribed using the StrataScript QPCR cdna synthesis kit, using our QPCR-grade, RNase H minus StrataScript RT in just 15 minutes. The cdna of interest is then quantified using the second real-time PCR module, using either our Brilliant or FullVelocity SYBR QPCR Master Mixes b. Our Brilliant 2-step QRT-PCR Master Mixes b are designed for the highest sensitivity, while our FullVelocity SYBR Green 2-Step QRT-PCR Master Mix b is ideal for speed and economy. 1 3 pg RNA 40 Company B (RSq ) 0.998; Eff. = 89.9% Company Q (RSq ) 0.998; Eff. = 84.8% Company I (RSq ) 0.997; Eff. = 83.4% 0.1 PANEL A Ct (drn) 30 Fluorescence (drn) 10 ng 0.1 ng StrataScript QPCR cdna Synthesis Kit (RSq ) 0.998; Eff. = 85.2% Initial Quantity (copies) PANEL B Cycles StrataScript RT Competitor Figure 7 ACHIEVE THE HIGHEST QRT-PCR SENSITIVITY USING A TWO-STEP QRT-PCR FORMAT Using first-strand cdna generated according to each manufacturer's recommended protocol, we performed real-time quantification of GDH, a medium abundance RNA target, using our Brilliant SYBR Green 2-Step QRT-PCR Master Mix. Our two-step kit was far more sensitive and linear than competitor kits, delivering an earlier Ct value across a wider range of RNA input (3 µg to 3 pg). Other kits (Company B, Q, and I) failed to produce cdna from 3 pg of RNA (red circle). Figure 8 MORE SENSITIVE RESULTS USING THE STRATASCRIPT RT IN REAL-TIME ANALYSIS Our results (in blue) show that using the StrataScript RT, which is RNase H negative, with our Brilliant 2x QPCR Master Mix in real-time analysis resulted in a lower Ct and more sensitive detection of GUS, compared to a competitor's RNase H+ RT (in red). (Mx3000P QPCR System)

9 UNIVERSAL QPCR TOTAL RNAs > STRATAGENE QPCR REFERENCE TOTAL RNAs QPCR-Grade Reference RNA, DNA-Free Stratagene QPCR Reference Total RNAs are high-quality, universal RNA controls for quantitative PCR gene-expression analysis. In order to reliably compare data across multiple experiments and instruments, it is essential to have a constant reference material to assess the performance of each QPCR reaction and quantify gene expression levels. Maximal Gene Representation The Stratagene QPCR Reference Total RNAs are used to directly compare data between multiple experiments (Figure 9). These RNAs are extracted from quality cell lines representing different human and mouse tissues, pooled together for maximal gene representation. The broad gene coverage allows you to use the RNA reference material as a universal reference for nearly any human or mouse gene being investigated in QPCR experiments. These high-quality human or mouse total RNA controls are manufactured for maximum representation of low, medium, and high-abundant gene transcripts. Our QPCR reference total RNAs are qualified for QPCR and guaranteed to be free of genomic DNA. We manufacture these control RNAs at industrial scale lot sizes and perform stringent quality-controls to maximize lot-to-lot reproducibility. We offer QPCR reference total RNA from both human and mouse cells. The QPCR Human Reference Total RNA is a collection of RNA pooled from 10 human cell lines derived from different tissues. The QPCR Mouse Reference Total RNA is derived from RNA pooled from 11 mouse cell lines which are also derived from tissues. We choose cell lines, rather than tissues, as our starting material since we have found that this is the most consistent and highest quality source of RNA. Stratagene QPCR Human Reference RNA (QHRR) Experiment #1 Experiment #2 QHRR Primers and/or Probes (Gene of Interest + Endogenous Control) Treated Sample Untreated Sample QHRR Real-Time QRT-PCR Primers and/or Probes (Endogenous Control) QHRR Primers and/or Probes (Gene of Interest + Endogenous Control) Treated Sample Untreated Sample Real-Time QRT-PCR Normalize Samples to QHRR Directly Compare Data Directly Compare Data Normalize Samples to QHRR Figure 9 ACCURATELY MEASURE RELATIVE EXPRESSION LEVELS AND COMPARE DATA FROM MULTIPLE EXPERIMENTS Use the Stratagene QPCR Reference Total RNA to measure endogenous control levels alongside your treated and untreated samples. Normalize samples to the endogenous control to determine relative expression levels of your gene of interest.

10 8>9 QPCR AND QRT-PCR KITS > BRILLIANT AND FULLVELOCITY QPCR AND QRT-PCR REAGENTS Total Reagent Solutions for Sensitive QPCR of Two to Four Targets Our versatile Brilliant QPCR and QRT-PCR reagents (Table 4) provide a highly sensitive solution for real-time PCR detection and gene quantification. Our broad Brilliant product portfolio covers every real-time experience level from novice to expert, with a choice of convenient Taq-based master mixes or core reagent kits for assay optimization. The FullVelocity SYBR QPCR and QRT-PCR Master Mixes use a novel, non-taq enzyme designed for high-speed, economical results. Our Brilliant SideStep Kits allow you to go from cells to real-time QPCR while avoiding nucleic acid purification steps and associated sample loss. Highly Sensitive Detection of DNA or RNA using SYBR Green Our Brilliant SYBR Green QPCR and QRT-PCR reagents provide a universal solution to real-time QPCR detection and gene quantification, and exhibit greater sensitivity compared to other SYBR Green kits. SYBR Green dye binds to any PCR product, and therefore does not require the use of sequencespecific probes. All Brilliant reagent kits contain SureStart Taq DNA Polymerase, a hot-start version of Taq that minimizes amplification of non-specific PCR products. Sensitive and Specific Probe-based Detection of up to Two Targets Brilliant probe-based QPCR and QRT-PCR b,c reagents are compatible with sequence-specific probes including TaqMan probes, Molecular Beacons, and Scorpions. These reagents offer a wide linear dynamic range of amplification (Figure 10). Our QRT-PCR kits contain StrataScript reverse transcriptase and are available in one-step and two-step formats. A passive reference dye is included for versatility and to maximize performance on different instrument platforms. REAL-TIME QPCR AND QRT-PCR REAGENTS GUIDE Format Master Mix SYBR GREEN DETECTION DNA (cdna) Quantification Brilliant SYBR Green QPCR Master Mix High Speed FullVelocity SYBR Master Mix Green QPCR Master Mix Core Reagent Kit (Standard dntps) RNA/DNA Preparation and Amplification Brilliant SYBR Green Core Reagent Kit Brilliant SYBR Green SideStep QPCR Master Mix 1-Step Brilliant SYBR Green QRT-PCR Master Mix, 1-Step FullVelocity SYBR Green QRT-PCR Master Mix, 1-Step RNA Quantification 2-Step Brilliant SYBR Green QRT-PCR Master Mix, 2-Step FullVelocity SYBR Green QRT-PCR Master Mix, 2-Step StrataScript QPCR cdna Synthesis Kit plus Brilliant SYBR Green Core Reagent Kit Brilliant SYBR Green SideStep QRT-PCR Master Mix Format Master Mix Core Reagent Kit (Standard dntps) RNA Preparation and Amplification PROBE-BASED DETECTION DNA (cdna) Quantification Brilliant QPCR Master Mix (up to 2 targets) or Brilliant Multiplex QPCR Master Mix (up to 4 targets) Brilliant QPCR Core Reagent Kit 1-Step Brilliant QRT-PCR Master Mix, 1-Step Brilliant QRT-PCR Core Reagent Kit, 1-Step Brilliant SideStep QRT-PCR Master Mix RNA Quantification PANEL A PANEL B 2-Step Brilliant QRT-PCR Master Mix, 2-Step Stratascript QPCR cdna Synthesis Kit plus Brilliant QPCR Core Reagent Kit Table 4 CHOOSE THE RIGHT MASTER MIX OR CORE REAGENT DEPENDING ON YOUR APPLICATION

11 Multiplex up to Four Simultaneous Targets The Brilliant Multiplex QPCR Master Mix b,c allows you to amplify up to four targets in a single reaction. Each multiplex reaction requires far less template than if the targets were analyzed in separate reactions. This is especially useful for rare or limited samples, such as those obtained from biopsies or laser micro-dissection (LMD). Moreover, the sensitivity remains equivalent to that seen in singleplex (Figure 11). Thus, you maximize the use of each precious sample without sacrificing sensitivity. The Brilliant multiplex QPCR master mix also provides sufficient reaction components to accurately quantify both low- and high-abundance targets in the same tube. This allows you to more successfully multiplex without concern for bias due to abundance level. In addition, you will save money when you use the Brilliant multiplex QPCR master mix versus running each target in a single reaction. QPCR and QRT-PCR Directly from Cells Without Purification Our Brilliant SYBR Green SideStep QPCR and QRT-PCR Reagents a,b take advantage of our new SideStep buffer technology to ensure accurate gene expression data by minimizing the loss or degradation of nucleic acid molecules by going directly from cells to a real-time QPCR amplification. Fast and Economical Detection of DNA or RNA using SYBR Green Our FullVelocity SYBR Green QPCR and QRT-PCR Master Mixes b use the novel FullVelocity DNA polymerase to provide rapid and efficient real-time quantification of DNA or cdna. Using a fast cycling protocol with a combined annealing/extension step, the polymerase allows 40-cycle amplification to be completed in less than one hour on most real-time instrument platforms. In addition, the FullVelocity DNA polymerase is less prone to inhibition by SYBR Green I than Taq DNA polymerase, promoting greater efficiency. Since our master mixes provide rapid and efficient quantification of RNA in a convenient format, they are the ideal choice when reproducibility and throughput are of primary concern. Figure 10 Figure 4 THE BRILLIANT QPCR MASTER MIX DELIVERS 10 ORDERS OF FIGURE NOTES HEADS/TITLES MAGNA ALIQUAM ERAT MAGNITUDE DYNAMIC RANGE Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod We amplified a plasmid target using the Brilliant QPCR Master Mix on our Mx3000P tincidunt ut laore het dolore magna al iquam erat volutpat. Ut wisi enim ad minim veniam, quis QPCR System and detected with a TaqMan probe. A standard curve showed a dynamic nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliqu ip ex ea commodo conseat.as detection range from 3x10 10 to 3 copies. ed diam nonummy nibh euismod tinci dolore magna aliquam erat volutpat. Figure 11 EQUIVALENT PERFORMANCE WITH FOUR-TARGET MULTIPLEX USING BRILLIANT MULTIPLEX QPCR MASTER MIX We amplified four targets enos (FAM ), HFE (HEX ), CFTR (ROX ), and Cyclophilin (Cy 5) in singleplex and multiplex using the Brilliant Multiplex QPCR Master Mix on the Mx3000P QPCR System. Ct values for each target were virtually identical in the two reactions, indicating full sensitivity and performance in multiplex.

12 10>11 QPCR INSTRUMENTATION & ANALYSIS SOFTWARE > MX3000P AND MX3005P QPCR SYSTEMS The Highest Performance and Flexibility in QPCR Systems at an Affordable Price With the Mx3000P QPCR System e, we were the first to offer an affordable system that supported multiple QPCR applications. The new Mx3005P QPCR System e contains additional enhancements to support an even wider range of QPCR applications. Features of the Mx3000P QPCR System The Mx3000P System is a high-performance, full-featured instrument system designed to accommodate basic experimental design and offer the flexibility required for more advanced applications (Figure 12). Mx3000P QPCR System Four optical channels with user-selected filters for greater flexibility Broad wavelength range excitation supports most fluorescent dyes Single photomultiplier tube (PMT) for detection ensures superior sensitivity and linear dynamic range to 10 orders of magnitude Features of the Mx3005P QPCR System The Mx3005P QPCR System expands on the Mx3000P system by offering unmatched flexibility and capability to support even more real-time QPCR applications and chemistries. The system features five customer-selected filters and a custom filter path feature to support detection of FRET-based fluorescence. The enhanced features of the Mx3005P system will accommodate your research needs now and in the future (Figure 13). Mx3005P QPCR System Five optical channels with user-selected filters for greater flexibility Defined excitation and emission detection wavelengths are ideal for superior multiplex results, up to five targets simultaneously (Figure 14) Open platform design supports all fluorescent chemistries and numerous applications Figure 12 THE Mx3000P QPCR SYSTEM Four-color, 96-well format fully featured QPCR system. [Also includes MxPro QPCR Software.] Figure 13 THE Mx3005P QPCR SYSTEM Five-color, 96-well format, open platform QPCR system with independent filter wheel control for detection of FRET-based fluorescence. [Also includes MxPro QPCR Software.]

13 Advanced Optical System The Mx optical system was the first design to incorporate narrow wavelength bandpass filters for excitation and emission with scanning fiber optics. The photomultiplier tube (PMT) has a large dynamic range of detection and a low signal-to-noise ratio, allowing low- to high-abundance targets to be accurately quantified (Figure 15). The scanning motion system of the fiber optic cable eliminates optical variation based on well position in the 96-well block, thus eliminating the need for signal correction by calibration or reference dyes. Precision Thermal System The 96-well thermal system of the Mx3000P and Mx3005P systems uses a Peltier-based design. The design ensures uniform thermal accuracy for amplification reproducibility from well to well and run to run. Powerful Data Analysis Software The MxPro QPCR Software for the Mx3000P and Mx3005P systems combines cutting-edge data analysis algorithms with intuitive organization designed for ultimate ease of use. The data analysis module includes two automated methods for baseline subtraction and threshold setting, both of which can be customized for any particular application. The Comparative Quantification module automatically determines gene expression fold change, calculates statistical error, and generates publication quality charts (Figure 16). The MxPro QPCR software is also capable of exporting all plots, charts, and tables to Microsoft Excel,.xml, and.txt formats, and Microsoft PowerPoint. MxPro QPCR Software Intuitive organization and easy to use View and analyze data in real time Multiple customizable data analysis algorithms for baseline correction and threshold setting Export images directly from the software, export the raw data to re-create the image, and export the text data in multiple formats Figure 14 FIVE TARGET MULTIPLEX STANDARD CURVES Three replicates of four-fold dilutions of QPCR Human Universal Reference RNA detecting five gene targets simultaneously. Detection from the highest abundance to the lowest abundance gene target (CYCLO to ENOS) covers a Ct range from Figure 15 Figure 4 THE Mx OPTICAL SYSTEM DESIGN FIGURE NOTES HEADS/TITLES MAGNA ALIQUAM ERAT The halogen lamp in the instrument systems provides a wide range of excitation, allowing Lorem more ipsum dye dolor flexibility sit amet, with consectetuer more intensity adipiscing than standard elit, sed light diam emitting nonummy diodes nibh euismod (LEDs). tincidunt The ut excitation laore het and dolore emission magna filters al iquam are defined erat volutpat. to narrow Ut wisi wavelengths enim ad to minim veniam, quis minimize nostrud fluorescence exerci tation signal ullamcorper crosstalk. suscipit Fiber lobortis optic bundles nisl ut aliqu channel ip ex the ea light commodo into conseat.as the ed plate diam and nonummy back to nibh the PMT euismod to ensure tinci dolore minimal magna signal aliquam loss. erat volutpat. Figure 16 MxPRO QPCR SOFTWARE GENE EXPRESSION DATA The MxPro Software automatically calculates and displays fold expression change. Data is displayed as normalized fold gene expression to a reference control on a chart using a base 2 logarithmic scale with upper and lower error limits based on variation in the replicates. In the figure above, fold expression change for six genes across two treatments is displayed.

14 12>13 Ordering Information PRODUCT QUANTITY / FORMAT CATALOG NO. Sample Preparation FOR DIRECT TO QRT-PCR OR QPCR FROM CELL LYSATES Brilliant SideStep QRT-PCR Master Mix, 1-step 400 rxn Brilliant SYBR Green SideStep QPCR Master Mix 400 rxn Brilliant SYBR Green SideStep QRT-PCR Master Mix, 2-Step 400 rxn SideStep Lysis and Stabilization Buffer 5 ml ml SideStep QPCR cdna Synthesis Kit 50 rxn TOTAL RNA PURIFICATION FROM PARAFFIN-EMBEDDED TISSUES Absolutely RNA FFPE Kit 50 preps Absolutely RNA FFPE Kit (w/o deparaffinization reagents) 50 preps HIGHLY PURE TOTAL RNA Absolutely RNA Microprep Kit 50 preps Absolutely RNA 96 Microprep Kit 2 plates plates Absolutely RNA Miniprep Kit 50 preps Absolutely RNA Nanoprep Kit 50 preps MESSENGER RNA ISOLATION Absolutely mrna Purification Kit 10 preps SideStep mrna Enrichment Kit 10 preps cdna Synthesis for Two-Step QRT-PCR StrataScript QPCR cdna Synthesis Kit 50 rxn StrataScript Reverse Transcriptase (50 U/µl) 10,000 units (200 U/µl) 10,000 units Universal QPCR Control RNAs Stratagene QPCR Reference Total RNA, Human 25 µg Stratagene QPCR Reference Total RNA, Mouse 25 µg QPCR Systems Mx3000P QPCR System 4-color system (110v) with notebook computer color system (110v) with desktop computer color system (230v) with notebook computer color system (230v) with desktop computer Mx3005P QPCR System 5-color system (110v) with desktop computer color system (110v) with notebook computer color system (230v) with desktop computer color system (230v) with notebook computer QPCR and QRT-PCR Reagents Brilliant Multiplex QPCR Master Mix Probe-based detection 200 rxn x 25 µl Brilliant QPCR Master Mix Kits SYBR Green-based detection 400 rxn x 25 µl Probe-based detection 400 rxn x 25 µl Brilliant QPCR Core Reagent Kits SYBR Green-based detection 400 rxn x 25 µl Probe-based detection 400 rxn x 25 µl Brilliant QRT-PCR Core Reagent Kit, 1-step Probe-based detection 400 rxn x 25 µl Brilliant QRT-PCR Master Mix Kits, 1-step SYBR Green-based detection 400 rxn x 25 µl Probe-based detection 400 rxn x 25 µl

15 PRODUCT QUANTITY / FORMAT CATALOG NO. Brilliant QRT-PCR Master Mix, 2-Step Probe-based detection 400 rxn x 25 µl SYBR Green-based detection 400 rxn x 25 µl Brilliant SideStep QPCR Master Mix SYBR Green-based detection 400 rxn x 25 µl Brilliant SideStep QRT-PCR Master Mix, 1-step SYBR Green-based detection 400 rxn x 25 µl Brilliant SideStep QRT-PCR Master Mix, 2-Step SYBR Green-based detection 400 rxn x 25 µl FullVelocity QPCR Master Mix Kits SYBR Green-based detection 400 rxn x 25 µl FullVelocity QRT-PCR Master Mix Kits, 1-step SYBR Green-based detection 400 rxn x 25 µl FullVelocity QRT-PCR Master Mix, 2-Step SYBR Green-based detection 400 rxn x 25 µl LEGAL LANGUAGE Absolutely mrna, SideStep, Mx3005P, MxPro, and FullVelocity are trademarks of Stratagene in the United States. Absolutely RNA, Brilliant, Mx3000P, and StrataScript are registered trademarks of Stratagene in the United States. Agilent is a registered trademark of Agilent Technologies. Excel and PowerPoint are registered trademarks of the Microsoft Corporation. SYBR is a registered trademark of Molecular Probes. Scorpions is a trademark of DxS Ltd. TaqMan is a registered trademark of Roche Molecular Systems, Inc. TRI Reagent is a registered trademark of Molecular Research Center, Inc. FAM, HEX, and ROX are trademarks of Applera Corporation or its subsidiaries in the US and certain other countries. Cy 3 and Cy 5 are trademarks of Amersham Biosciences. a. Patents Pending b. Purchase of this product is accompanied by a license under the foreign counterparts of U.S. Patents Nos. 4,683,202, 4,683,195 and 4,965,188 for use in the polymerase chain reaction (PCR) process, where such process is covered by patents, in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Applied Biosystems or as purchased, i.e., an authorized thermal cycler. c. Use of labeling reagents may require licenses from entities other than Stratagene. For example, use of fluorogenic probes in 5' nuclease assays may require licenses under U.S. Patent Nos. 6,214,979, 5,804,375, 5,210,015 and 5,487,972 owned by Roche Molecular Systems, Inc. and under U.S. Patent No. 5,538,848 owned by Applied Biosystems. TaqMan is a registered trademark of Roche Molecular Systems, Inc d. Purchase of this PCR-related product does not convey any rights under the foreign counterparts of the PCR patents owned by Roche Molecular Systems. A license to use the PCR process, where such process is covered by patents, accompanies the purchase of certain reagents from Stratagene when used in conjunction with an Authorized Thermal Cycler. e. Purchase of this product is accompanied by a license under the foreign counterparts of U.S. Patent Nos. 4,683,195, 4,683,202 and 4,965,188 covering the Polymerase Chain Reaction ( PCR ) process, where such process is covered by patents. This instrument is an Authorized Thermal Cycler for use with applications licenses available from Applied Biosystems. Its use with Authorized Reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents.

16 AMPLIFICATION CELL BIOLOGY CLONING MICROARRAYS NUCLEIC ACID ANALYSIS PROTEIN FUNCTION & ANALYSIS QUANTITATIVE PCR SOFTWARE SOLUTIONS Stratagene USA and Canada Order: x3 Technical Services: x2 Stratagene Europe Order: Technical Services: Stratagene Japan K.K. Order: Technical Services: Distributors > For a list of worldwide distributors, please visit our website North Torrey Pines Road La Jolla, CA USA BR68