Gene Expression

Gene Expression Analysis Solutions

Gene Expression Workflow

Successful gene expression analysis using real-time PCR

requires critical evaluation of each step in the workflow

to ensure that accurate, reproducible, publishable data

are produced. Bio-Rad Laboratories, Inc. offers an array

of tools that assists with successful data generation and

analysis, reducing your time to results.

■■ Aurum™ total RNA mini kit

■■ Aurum total RNA 96 kit

■■ Aurum total RNA fatty and fibrous tissue kit

■■ iScript™ reverse transcription supermix for RT-qPCR

■■ iQ™ SYBR® Green supermix

■■ SsoFast™ EvaGreen® supermix

■■ SsoFast probes supermix

■■ Experion™ automated electrophoresis system

Quality Assessment

Reverse Transcription and Real-Time PCR Reagents

SamplePreparation

■■ CFX384 Touch™ real-time PCR detection system

■■ CFX96 Touch™ real-time PCR detection system

■■ CFX Connect™ real-time PCR detection system

■■ MiniOpticon™ real-time PCR detection system

■■ CFX Manager™ software■■ qbasePLUS software■■ Precision Melt Analysis™ software

Real-Time Data Collection

Data Analysis and Optimization

Aurum Total RNA 96 Kit

Experion Automated Electrophoresis System

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Electropherogram of high-quality eukaryotic total RNA. The clearly defined peaks and minimal background fluorescence indicate intact, undegraded RNA.

Using the Experion system, an RNA quality indicator (RQI; a number on a scale of 1–10) can be automatically generated for mammalian total RNA samples. An RQI of 1 indicates highly degraded RNA, while an RQI of 10 indicates highly intact RNA.

Sample PreparationObtaining high-quality, contaminant-free RNA rapidly and efficiently is a crucial first step for most gene expression experiments.

Aurum total RNA kits are used to isolate high-quality, high-yield, DNA-free total RNA from cultured cells, bacteria, yeast, and plant and animal tissues. The kits utilize a stringent reagent containing guanidine isothiocyanate and ß-mercaptoethanol for efficient lysis and quick RNase inactivation, followed by purification on a silica membrane. The entire purification process, in column and 96-well plate formats, is completed in less than 60 min. Easy-to-use spin- and vacuum-compatible protocols are available.

The Aurum total RNA fatty and fibrous tissue kit offers a solution for difficult-to-disrupt samples rich in RNases.

DNase I is included with all Bio-Rad RNA purification kits for complete digestion of genomic DNA.

Quality AssessmentUsing RNA of high purity and integrity in quantitative PCR experiments is essential to obtain meaningful results. RNA integrity is commonly assessed by visual inspection of RNA bands on ethidium bromide–stained agarose gels following electrophoresis. However, this method is subjective and requires relatively large quantities of starting material. The introduction of microfluidic electrophoresis systems has made the determination of RNA integrity effortless, unbiased, and accessible to non-experienced users requiring only minute amounts of sample.

Bio-Rad’s Experion automated electrophoresis station performs all of the steps of gel-based electrophoresis in one compact unit. It’s an electrophoresis cell, power supply, and imager built into a single device designed for easy operation. A highly accurate laser provides precise fluorescence detection. It automates analysis by combining electrophoresis, staining, band detection, imaging, and analysis in a 30 min step.

Gene Expression Analysis Solutions

Sso7d Polymerase

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Real-Time Data CollectionBio-Rad’s real-time PCR systems are designed to bring outstanding thermal performance and sensitive optical detection to all of your real-time PCR applications. By pairing proven real-time instruments with quality PCR reagents and powerful analysis software, you can consistently obtain high-quality data to fuel your research discoveries.

CFX384 Touch Real-Time PCR Detection System

CFX96 Touch Real-Time PCR Detection System

Feature MiniOpticon System CFX Connect System CFX96 Touch System CFX384 Touch System

Capacity 48 wells 96 wells 96 wells 384 wells

Light source 48 blue-green light-emitting 3 LEDs in optics shuttle 6 LEDs in optics shuttle 5 LEDs in optics shuttle diodes (LEDs)

Optical detection 2 photodiodes 3 photodiodes 6 photodiodes 5 photodiodes

Excitation range 470–500 nm 450–535 nm 450–684 nm 450–650 nm

Detection range 523–700 nm 515–580 nm 515–730 nm 515–690 nm

Multiplex capability Up to 2 targets Up to 2 targets Up to 5 targets Up to 4 targets

Maximum gradient span 16°C 24°C 24°C 24°C

Maximum ramp rate 2.5°C/sec 5°C/sec 5°C/sec 2.5°C/sec

Real-time PCR software CFX Manager software CFX Manager software CFX Manager software CFX Manager software

Reverse Transcription and Real-Time PCR ReagentsOptimal gene expression analysis results rely on the sensitivity and efficiency of both reverse transcription and qPCR steps. The iScript reverse transcription supermix for RT-qPCR is a simple, fast, and sensitive first-strand cDNA synthesis kit for gene expression analysis using real-time qPCR. In one tube, the preblended 5x supermix contains all the necessary components except RNA template for reverse transcription. The 5x supermix includes a blend of oligo(dT) and random primers for unbiased representation of 5' and 3' regions of the target gene. The reverse transcriptase is derived from Moloney murine leukemia virus (MMLV) and contains the RNase H+ component for greater qPCR sensitivity. iScript one-step RT-PCR kits are available for SYBR® Green and probe-based detection.

Bio-Rad’s SsoFast qPCR supermixes are based on patented* Sso7d fusion protein technology. A DNA-binding domain, Sso7D from Sulfolobus solfataricus, is fused to a hot-start DNA polymerase to provide enhanced processivity, speed, and tolerance to PCR inhibitors. Sso7d fusion polymerase allows instant polymerase activation and rapid polymerization kinetics for faster qPCR results. SsoFast supermixes (for dye-based and probe-based detection) are formulated to provide maximum efficiency, sensitivity, and reproducibility while allowing fast thermal cycling conditions.

* U.S. patents 6,627,424; 7,541,170; and 7,560,260.

The unique fusion polymerase in SsoFast EvaGreen supermix delivers extreme speed and generates exceptional qPCR results in less than 30 min. Tenfold serial dilutions of 10 ng to 100 ag of cDNA from human spleen were used in each 20 μl reaction to detect 18S rRNA. 18S rRNA efficiency = 101.8%, r = 0.997. Total qPCR run time = 29 min. RFU, relative fluorescence units.

CFX Automation System

In laboratories where processing large volumes of plates is required, unattended operation is the solution. The CFX automation system integrates with the CFX96 Touch and CFX384 Touch real-time PCR detection systems, enabling up to 20-plate runs for high-throughput operation.

CFX Automation System

Feature MiniOpticon System CFX Connect System CFX96 Touch System CFX384 Touch System

Capacity 48 wells 96 wells 96 wells 384 wells

Light source 48 blue-green light-emitting 3 LEDs in optics shuttle 6 LEDs in optics shuttle 5 LEDs in optics shuttle diodes (LEDs)

Optical detection 2 photodiodes 3 photodiodes 6 photodiodes 5 photodiodes

Excitation range 470–500 nm 450–535 nm 450–684 nm 450–650 nm

Detection range 523–700 nm 515–580 nm 515–730 nm 515–690 nm

Multiplex capability Up to 2 targets Up to 2 targets Up to 5 targets Up to 4 targets

Maximum gradient span 16°C 24°C 24°C 24°C

Maximum ramp rate 2.5°C/sec 5°C/sec 5°C/sec 2.5°C/sec

Real-time PCR software CFX Manager software CFX Manager software CFX Manager software CFX Manager software

CFX96 Touch and CFX384 Touch Systems

The CFX96™ and CFX384™ optical reaction modules build on the power and flexibility of the C1000 Touch™ thermal cycler to create exceptional real-time PCR detection systems.

■■ Reliable performance — systems use long-lasting solid-state technology with LEDs and photodiodes for precise quantification and target discrimination

■■ Low sample and reagent volumes — 4- and 5-target multiplex experiments yield optimal results, using sample volumes as low as 3 μl (CFX384 Touch system)

■■ Unparalleled stand-alone functionality without a computer — quickly set up runs and monitor amplification traces in real time on the integrated LCD touch screen

■■ Data analysis when and where you want — email notification with an attached data file can be sent when a run is finished

CFX Connect System

The CFX Connect system offers two-target analysis with the same reliable performance as the CFX96 Touch system.

■■ Easy startup — obtain great results right away with factory-calibrated dyes, quick setup, and intuitive software

■■ Effortless optimization — save time and reduce costs by optimizing assays in a single run using the thermal gradient

■■ Powerful data analysis tools — accurately and quickly validate and analyze data with the advanced analysis modules of CFX Manager software

* U.S. patent 7,632,464.

The patented* reduced-mass sample block in the CFX96 Touch system heats and cools more quickly than standard blocks, so average ramp rates are increased and overall run times are reduced.

As the CFX96 Touch system optics shuttle travels across the plate, light is focused directly into the center of each well, resulting in high sensitivity and no cross talk.

MiniOpticon System

The compact MiniOpticon system places the most advanced real-time PCR applications and gel-free PCR analysis techniques within the reach of every researcher.

■■ Optimal temperature control — based on the MJ Mini™ thermal cycler, the MiniOpticon system boasts precise thermal control and a powerful thermal gradient feature

■■ Precise optics — a fixed array of 48 LEDs enables reproducible and accurate multiplexing capability of up to 2 targets

■■ Compact footprint — the MiniOpticon system is one of the smallest and most portable systems available for real-time PCR detection

Data Analysis and OptimizationCFX Manager Software

CFX Manager software is powerful software for a powerful instrument.

■■ Intuitive software for fast time to results — features a startup wizard and express loading of commonly used plates and protocols

■■ Advanced gene expression analysis options — include normalized gene expression determination using multiple reference genes and individual reaction efficiencies

■■ Gene Study feature — confidently combine runs and analyze results generated from multiple plates

■■ Multiple editions — Security, and Chinese and Russian language versions are available

■■ Faster decision making — easily visualize all your important data in a single window using Custom Data View

qbasePLUS Software

Biogazelle and Bio-Rad have teamed up to offer a powerful suite of tools that speeds up real-time PCR data analysis and accelerates discovery. A premium license for qbasePLUS software is included with each CFX Connect, CFX96 Touch, and CFX384 Touch real-time PCR detection system.

■■ Wizard-guided statistical analysis

■■ Greater confidence in results and manuscript acceptance through inclusion of experimental annotations and RDML output, thereby assisting with MIQE compliance

■■ Inter-lab collaboration facilitated by importing and comparing data from any real-time PCR platform

■■ Advanced quality controls, normalization, and inter-run calibration for improved data reliability

■■ Windows, Macintosh, and Linux compatible

Streamline assessment of run results using CFX Manager software data analysis module (back) and gene expression analysis module (front).

Thermal gradient experiment for optimizing annealing temperature. A tenfold dilution series (106 to 10 copies) of plasmid containing GAPDH was amplified in the presence of SYBR® Green dye using a protocol with annealing temperatures ranging from 55 to 68°C. Results are presented for three temperatures, showing 62°C as the optimal annealing temperature in this case, with early Cq values and the highest standard curve efficiency. Cq, quantification cycle; RFU, relative fluorescence units.

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— SYBR® Green: E = 68.3%

— SYBR® Green: E = 98.7%

— SYBR® Green: E = 93.6%

Life ScienceGroup

11-0290 0611 Sig 0211Bulletin 5990 Rev C US/EG

Bio-Rad Laboratories, Inc.

Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 31 3689 6600 Canada 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Malaysia 60 3 2117 5260 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3170 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 Thailand 66 2 6518311 United Kingdom 020 8328 2000

Ordering InformationCatalog # Description

Real-Time PCR Detection Systems185-5196 CFX96 Touch Real-Time PCR Detection System,

includes C1000 Touch thermal cycler chassis, CFX96 optical reaction module, CFX Manager software, license for qbasePLUS software, communication cable, reagents, consumables

185-5484 CFX384 Touch Real-Time PCR Detection System, includes C1000 Touch thermal cycler chassis, CFX384 optical reaction module, CFX Manager software, license for qbasePLUS software, communication cable, reagents, consumables

185-5200 CFX Connect Real-Time PCR Detection System, includes CFX Connect thermal cycler chassis, CFX Connect optical reaction module, CFX Manager software, license for qbasePLUS software, communication cable, reagents, consumables

CFB-3120 MiniOpticon Two-Color Real-Time PCR Detection System, includes optical housing, MJ Mini thermal cycler, analysis software

184-5001 CFX Manager Software, Security Edition, includes 1 user license, installation CD, HASP HL key

184-5008 CFX Manager Software, Chinese Edition, includes 3 user licenses, installation CD, 3 HASP HL keys

184-5028 CFX Manager Software, Russian Edition, includes 3 user licenses, installation CD, 3 HASP HL keys

184-5025 Precision Melt Analysis Software, includes 2 user licenses, installation CD, 2 HASP HL keys, melt calibration kit

184-5072 CFX Automation System, includes robotic plate handler, base tray, bar code scanner, CFX automation control software CD

Accessories for CFX Connect, CFX96 Touch, and CFX384 Touch SystemsHSP-9601 Hard-Shell® Thin-Wall 96-Well Skirted PCR Plates,

white shell, clear well, 50HSP-3805 Hard-Shell Thin-Wall 384-Well Skirted PCR Plates,

clear shell, white well, 50MSB-1001 Microseal® 'B' Adhesive Seals, optically clear, 100TCS-0803 Optical Flat 8-Cap Strips, for 0.2 ml PCR tubes and

plates, ultraclear, 120

Accessories for MiniOpticon SystemMLL-4851 Multiplate™ Low-Profile 48-Well Unskirted PCR

Plates, white, 50TCS-0803 Optical Flat 8-Cap Strips, for 0.2 ml PCR tubes and

plates, ultraclear, 120

Kits and Reagents732-6820 Aurum Total RNA Mini Kit, 50 preps, includes 50 RNA

binding columns, 50 capless collection tubes (2.0 ml), 100 capped sample tubes (2.0 ml), 50 capped sample tubes (1.5 ml), 1 vial lyophilized DNase I, RNase-free reagents, protocol overview

732-6800 Aurum Total RNA 96 Kit, 2 x 96-well preps, includes 2 growth membranes, sealing tape, 2 RNA binding plates, 2 collection microplates, 2 vials lyophilized DNase I, RNase-free reagents, protocol overview

732-6830 Aurum Total RNA Fatty and Fibrous Tissue Kit, 50 preps, includes 50 RNA binding columns, 50 capless collection tubes (2.0 ml), 100 capped sample tubes (2.0 ml), 50 capped sample tubes (1.5 ml), 1 vial lyophilized DNase I, RNase-free reagents and plasticware, 50 ml PureZOL™ RNA isolation reagent

Catalog # Description

Kits and Reagents (continued) 170-8898 iScript RT-qPCR Sample Preparation Reagent,

100 reactions, 10 ml, contains RNase inhibitors and RNA stabilizers

170-8841 iScript Reverse Transcription Supermix for RT-qPCR, 100 x 20 μl reactions, 4 x 100 μl of 5x supermix contains reverse transcriptase, RNase inhibitor, dNTPs, primers, MgCl2, stabilizers; 10 reactions of no-RT control supermix

172-5200 SsoFast EvaGreen Supermix, 200 x 20 μl reactions, 2x mix contains dNTPs, Sso7d fusion polymerase, MgCl2, EvaGreen dye, stabilizers

172-5230 SsoFast Probes Supermix, 200 x 20 μl reactions, 2x mix contains dNTPs, Sso7d fusion polymerase, MgCl2, stabilizers

172-5996 SsoFast EvaGreen Control Assay, predesigned kit for verifying performance of real-time PCR systems or training new users, includes template DNA, primers, supermix, water

170-8880 iQ™ SYBR® Green Supermix, 100 x 50 μl reactions, 2x supermix contains 100 mM KCl, 40 mM Tris-HCl, pH 8.4, 0.4 mM each dNTP (dATP, dCTP, dGTP, dTTP), 50 U/ml iTaq™ DNA polymerase, 6 mM MgCl2, SYBR® Green I, 20 nM fluorescein, stabilizers

Experion Automated Electrophoresis System700-7001 Experion System, 100–240 V, for RNA and DNA

analyses, includes electrophoresis station, priming station, vortex station, software, USB2 cable

EvaGreen is a trademark of Biotium, Inc. Bio-Rad Laboratories, Inc. is licensed by Biotium, Inc. to sell reagents containing EvaGreen dye for use in real-time PCR, for research purposes only. HASP is a trademark of Aladdin Knowledge Systems, Ltd. Linux is a trademark of Linus Torvalds. Macintosh is a trademark of Apple Inc. SYBR is a trademark of Molecular Probes, Inc. Bio-Rad Laboratories, Inc. is licensed by Molecular Probes, Inc. to sell reagents containing SYBR Green I for use in real-time PCR, for research purposes only. Windows is a trademark of Microsoft Corporation.

LabChip and the LabChip logo are trademarks of Caliper Life Sciences, Inc. Bio-Rad Laboratories, Inc. is licensed by Caliper Life Sciences, Inc. to sell products using the LabChip technology for research use only. These products are licensed under U.S. Patent Numbers 5,863,753; 5,658,751; 5,436,134; and 5,582,977; and pending patent

applications, and related foreign patents, for internal research and development use only in detecting, quantitating, and sizing macromolecules, in combination with microfluidics, where internal research and development use expressly excludes the use of this product for providing medical, diagnostic, or any other testing, analysis, or screening services, or providing clinical information or clinical analysis, in any event in return for compensation by an unrelated party.

Notice regarding Bio-Rad thermal cyclers and real-time systems: Purchase of this instrument conveys a limited non-transferable immunity from suit for the purchaser’s own internal research and development and for use in human in vitro diagnostics and all other applied fields under U.S. Patent Number 5,475,610 (Claims 1, 44, 158, 160–163, and 167 only), or corresponding claims in its non-U.S. counterpart, owned by Applera Corporation. No right is conveyed expressly, by implication, or by estoppel under any other patent claim, such as claims to apparatus, reagents, kits, or methods such as 5' nuclease methods. 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.

Bio-Rad’s real-time thermal cyclers are licensed real-time thermal cyclers under Applera’s U.S. Patent Number 6,814,934 B1 for use in research, human in vitro diagnostics, and all other fields except veterinary diagnostics.

Bio-Rad’s real-time thermal cyclers are covered by one or more of the following U.S. patents or their foreign counterparts owned by Eppendorf AG: U.S. Patent Numbers 6,767,512 and 7,074,367.

Practice of the patented 5' Nuclease Process requires a license from Applied Biosystems. The purchase of these products includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser’s own internal research when used with the separate purchase of Licensed Probe. No other patent rights are conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained from the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

Hard-Shell plates are covered by one or more of the following U.S. patents or their foreign counterparts owned by Eppendorf AG: U.S. Patent Numbers 7,347,977; 6,340,589; and 6,528,302.