Gene Expression Analysis Solutions. Gene Expression

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1 Gene Expression Analysis Solutions Gene Expression

Gene Expression Workflow Successful gene expression analysis using real-time PCR requires critical evaluation of

Reverse Transcription and Real-Time PCR Reagents iscript reverse transcription supermix for RT-qPCR iq SYBR Green

2 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. Reverse Transcription and Real-Time PCR Reagents iscript reverse transcription supermix for RT-qPCR iq SYBR Green supermix Quality Assessment Experion automated electrophoresis system SsoFast EvaGreen supermix SsoFast probes supermix Aurum total RNA mini kit Aurum total RNA 96 kit Sample Preparation Aurum total RNA fatty and fibrous tissue kit

3 Data Analysis and Optimization CFX Manager software qbase PLUS software Precision Melt Analysis software Real-Time Data Collection 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

Gene Expression Analysis Solutions Sample Preparation Obtaining high-quality, contaminant-free RNA rapidly and efficiently is a crucial first step for most gene expression experiments.

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

4 Gene Expression Analysis Solutions Sample Preparation Obtaining 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 Assessment Using 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. Fluorescence, relative S 28S Time, sec 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. Aurum Total RNA 96 Kit Experion Automated Electrophoresis System

Reverse Transcription and Real-Time PCR Reagents Optimal gene expression analysis results rely on the sensitivity and efficiency of both reverse

The iscript reverse transcription supermix for RT-qPCR is a simple, fast, and sensitive first-strand cdna synthesis kit for gene expression

In one tube, the preblended 5x supermix contains all the necessary components except RNA template for reverse transcription.

The reverse transcriptase is derived from Moloney murine leukemia virus (MMLV) and contains the RNase H+ component for greater qpcr sensitivity.

Real-Time Data Collection Bio-Rad s real-time PCR systems are designed to bring outstanding thermal performance and sensitive optical detection to

By pairing proven real-time instruments with quality PCR reagents and powerful analysis software, you can consistently obtain high-quality data to

A DNA-binding domain, Sso7D from Sulfolobus solfataricus, is fused to a hot-start DNA polymerase to provide enhanced processivity, speed, and

Sso7d fusion polymerase allows instant polymerase activation and rapid polymerization kinetics for faster qpcr results.

allowing fast thermal cycling conditions.

polymerase in SsoFast EvaGreen supermix delivers extreme speed and generates exceptional qpcr results in less than 30 min.

5 Reverse Transcription and Real-Time PCR Reagents Optimal 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. Real-Time Data Collection Bio-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. 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 CFX96 Touch Real-Time PCR Detection System RFU 10 3 CFX384 Touch Real-Time PCR Detection System Cycles 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 = Total qpcr run time = 29 min. RFU, relative fluorescence units. Sso7d Polymerase Feature MiniOpticon System CFX Connect System CFX96 Touch System Capacity 48 wells 96 wells 96 wells Light source 48 blue-green light-emitting 3 LEDs in optics shuttle 6 LEDs in optics shuttle diodes (LEDs) Optical detection 2 photodiodes 3 photodiodes 6 photodiodes Excitation range nm nm nm Detection range nm nm nm Multiplex capability Up to 2 targets Up to 2 targets Up to 5 targets Maximum gradient span 16 C 24 C 24 C Maximum ramp rate 2.5 C/sec 5 C/sec 5 C/sec Real-time PCR software CFX Manager software CFX Manager software CFX Manager software * U.S. patents 6,627,424; 7,541,170; and 7,560,260.

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

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

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 The

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

using the thermal gradient Powerful data analysis tools accurately and quickly validate and analyze data with the advanced analysis modules of CFX Manager software As the CFX96 Touch system optics

6 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 notification with an attached data file can be sent when a run is finished 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. CFX Automation System 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 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. 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. CFX384 Touch System 384 wells 5 LEDs in optics shuttle 5 photodiodes nm nm Up to 4 targets 24 C 2.5 C/sec CFX Manager software 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 * U.S. patent 7,632,464. 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 Optimization 67 C CFX Manager Software CFX Manager software is powerful software for a powerful instrument. RFU 10 4 10 3 10 2 Cq 40 35 30 25 SYBR Green: E = 68.

analysis options include normalized gene expression determination using multiple reference genes and individual reaction efficiencies RFU 10 4 10 3 10 2 Cq 30 25 20 SYBR Green: E = 98.

decision making easily visualize all your important data in a single window using Custom Data View 10 0 10 20 30 40 56 C 10 4 10 3 RFU Cq 10 2 10 0 10 20 30 40 15 30 25 20 15 1 2 3 4 5 6 SYBR

A tenfold dilution series (10 6 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.

Cq, quantification cycle; RFU, relative fluorescence units. Streamline assessment of run results using CFX Manager software data analysis module (back) and gene expression analysis module (front).

A premium license for qbase PLUS software is included with each CFX Connect, CFX96 Touch, and CFX384 Touch real-time PCR detection system.

7 Data Analysis and Optimization 67 C CFX Manager Software CFX Manager software is powerful software for a powerful instrument. RFU Cq SYBR Green: E = 68.3% Intuitive software for fast time to results features a startup wizard and express loading of commonly used plates and protocols C Advanced gene expression analysis options include normalized gene expression determination using multiple reference genes and individual reaction efficiencies RFU Cq SYBR Green: E = 98.7% 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 C RFU Cq SYBR Green: E = 93.6% Cycles log starting quantity Thermal gradient experiment for optimizing annealing temperature. A tenfold dilution series (10 6 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. Streamline assessment of run results using CFX Manager software data analysis module (back) and gene expression analysis module (front). qbase PLUS 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 qbase PLUS 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

8 Ordering Information Catalog # Description Real-Time PCR Detection Systems CFX96 Touch Real-Time PCR Detection System, includes C1000 Touch thermal cycler chassis, CFX96 optical reaction module, CFX Manager software, license for qbase PLUS software, communication cable, reagents, consumables CFX384 Touch Real-Time PCR Detection System, includes C1000 Touch thermal cycler chassis, CFX384 optical reaction module, CFX Manager software, license for qbase PLUS software, communication cable, reagents, consumables CFX Connect Real-Time PCR Detection System, includes CFX Connect thermal cycler chassis, CFX Connect optical reaction module, CFX Manager software, license for qbase PLUS software, communication cable, reagents, consumables CFB-3120 MiniOpticon Two-Color Real-Time PCR Detection System, includes optical housing, MJ Mini thermal cycler, analysis software CFX Manager Software, Security Edition, includes 1 user license, installation CD, HASP HL key CFX Manager Software, Chinese Edition, includes 3 user licenses, installation CD, 3 HASP HL keys CFX Manager Software, Russian Edition, includes 3 user licenses, installation CD, 3 HASP HL keys Precision Melt Analysis Software, includes 2 user licenses, installation CD, 2 HASP HL keys, melt calibration kit 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 Systems HSP-9601 Hard-Shell Thin-Wall 96-Well Skirted PCR Plates, white shell, clear well, 50 HSP-3805 Hard-Shell Thin-Wall 384-Well Skirted PCR Plates, clear shell, white well, 50 MSB-1001 Microseal 'B' Adhesive Seals, optically clear, 100 TCS-0803 Optical Flat 8-Cap Strips, for 0.2 ml PCR tubes and plates, ultraclear, 120 Accessories for MiniOpticon System MLL-4851 Multiplate Low-Profile 48-Well Unskirted PCR Plates, white, 50 TCS-0803 Optical Flat 8-Cap Strips, for 0.2 ml PCR tubes and plates, ultraclear, 120 Kits and Reagents 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 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 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) iscript RT-qPCR Sample Preparation Reagent, 100 reactions, 10 ml, contains RNase inhibitors and RNA stabilizers 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, MgCl 2, stabilizers; 10 reactions of no-rt control supermix SsoFast EvaGreen Supermix, 200 x 20 μl reactions, 2x mix contains dntps, Sso7d fusion polymerase, MgCl 2, EvaGreen dye, stabilizers SsoFast Probes Supermix, 200 x 20 μl reactions, 2x mix contains dntps, Sso7d fusion polymerase, MgCl 2, stabilizers SsoFast EvaGreen Control Assay, predesigned kit for verifying performance of real-time PCR systems or training new users, includes template DNA, primers, supermix, water 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 MgCl 2, SYBR Green I, 20 nm fluorescein, stabilizers Experion Automated Electrophoresis System Experion System, 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, , 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. Bio-Rad Laboratories, Inc. Life Science Group Web site USA Australia Austria Belgium Brazil Canada China Czech Republic Denmark Finland France Germany Greece Hong Kong Hungary India Israel Italy Japan Korea Malaysia Mexico The Netherlands New Zealand Norway Poland Portugal Russia Singapore South Africa Spain Sweden Switzerland Taiwan Thailand United Kingdom Bulletin 5990 Rev C US/EG Sig 0211

Real-Time PCR: CFX96 Touch System. CFX96 Touch Real-Time PCR Detection System

Real-Time PCR: CFX96 Touch System CFX96 Touch Real-Time PCR Detection System Advancing qpcr Together The CFX96 Touch real-time PCR detection system builds on the power and flexibility of the C00 Touch