Exceed the limit. SensiFAST Real-Time PCR Family. Superior Sensitive Quantification

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Griselda Hoover
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1 Exceed the limit SensiFAST Real-Time PCR Family Superior Sensitive Quantification

SensiFAST Real-Time PCR Kits Fast - optimized for fast cycling conditions (under 30 minutes), ideal for high-throughput High specificity - leading to better efficiency and more confidence in your

analysis techniques available and has become the standard in most laboratories.

2 SensiFAST Real-Time PCR Kits Fast - optimized for fast cycling conditions (under 30 minutes), ideal for high-throughput High specificity - leading to better efficiency and more confidence in your results Ultra-sensitive - perfect for low copy number samples Universal - compatible with all standard and fast cycling instruments Real-time PCR is one of the most powerful and sensitive gene analysis techniques available and has become the standard in most laboratories. Its effectiveness, particularly at amplification and quantification of low levels of nucleic acids, has driven the emergence of numerous applications, the main ones being gene expression analysis and pathogen detection/quantification. Other applications including cellular mrna and mirna quantification, biomarker discovery and validation, microarrays, microbial quantification, cancer risk assessment, gene dosage determination and detection of extremely low copy targets for forensic investigations. The increased use of real-time PCR has driven the demand for higher throughput and faster assays, to reduce the overall protocol time. This can be achieved by improving instruments, employing faster enzymes (without sacrificing accuracy) and shortening or even combining PCR steps. The new SensiFAST Kits have been specifically designed to meet these higher throughput needs, by combining a proprietary enzyme with antibody mediated hot-start and very rapid enzyme-extension rates with an industry leading and unique buffer chemistry. As a result, SensiFAST formulations provide the fastest cycling times while maintaining the high performance, reliability and high reproducibility of conventional real-time PCR reagents. SensiFAST real-time PCR kits can be used on all real-time instruments including the new generation of fast-cyclers. Exceed the limit with SensiFAST Real-Time PCR kits, available in configurations matched to all your applications and techniques, with both DNA and RNA templates.

Contents SensiFAST Kits 04 SensiFAST Real-Time PCR Reagents Selection Guide 04 SensiFAST SYBR Kits 06 SensiFAST Probe Kits 07 SensiFAST HRM Kit 08 SensiFAST Genotyping Kit 09 SensiFAST SYBR s 10

3 Contents SensiFAST Kits 04 SensiFAST Real-Time PCR Reagents Selection Guide 04 SensiFAST SYBR Kits 06 SensiFAST Probe Kits 07 SensiFAST HRM Kit 08 SensiFAST Genotyping Kit 09 SensiFAST SYBR s 10 SensiFAST Probe s 11 Technical Support For technical assistance or more information on these products, please contact us at tech@bioline.com or call us on INT: +44 (0) UK: +44 (0) DE: +49 (0) US: AUST: +61 (0)

4 SensiFAST Real-Time PCR Kits SensiFAST Real-Time PCR Reagents Selection Guide SensiFAST uses the latest discoveries and developments in real-time PCR to realize the fastest cycling times and greatest sensitivity, without compromising accuracy, reproducibility or performance. SensiFAST kits can be used on all real-time 4 SYBR Green Assays Probe Assays SYBR Green Assays HRM Probe Assays RNA Templates DNA/cDNA Templates SensiFAST SYBR No-ROX Kit SensiFAST SYBR Hi-ROX Kit SensiFAST SYBR Lo-ROX Kit SensiFAST SYBR & Fluorescein Kit SensiFAST Probe No-ROX Kit* SensiFAST Probe Hi-ROX Kit SensiFAST Probe Lo-ROX Kit SensiFAST Genotyping Kit SensiFAST HRM Kit SensiFAST SYBR No-ROX SensiFAST SYBR Hi-ROX SensiFAST SYBR Lo-ROX SensiFAST SYBR & Fluorescein SensiFAST Probe No-ROX * SensiFAST Probe Hi-ROX SensiFAST Probe Lo-ROX For more information please visit BioMark Fluidigm Thermal Cycler Dice (TP800) Takara Piko Real Thermo Eco Illumina SmartCycler Cepheid QuantStudio 12k Flex ViiA7 StepOne plus StepOne 7900HT FAST 7900HT FAST Applied Biosystems instruments and are also perfect for the new generation of fast-cyclers.

* Used for all instruments when multiplexing MX3005P MX3000P MX4000P Agilent (Stratagene) Xxpress BJS PrimeQ Quantica Techne Mastercycler ep realplex 2S Mastercycler ep realplex Eppendorf LightCycler

5 * Used for all instruments when multiplexing MX3005P MX3000P MX4000P Agilent (Stratagene) Xxpress BJS PrimeQ Quantica Techne Mastercycler ep realplex 2S Mastercycler ep realplex Eppendorf LightCycler Nano LightCycler 480 LightCycler 96 Roche CFX384 CFX96 MiniOpticon Chromo4 Opticon 2 Opticon iq 5 MyiQ icycler Bio-Rad Rotor-Gene Q Rotor-Gene 6000 Rotor-Gene 3000 Qiagen (Corbett) qtower Analytik Jena Use the selection table below to identify the most suitable product for your real-time PCR instrument... Recommended

SensiFAST SYBR Kits Features and Benefits: Accurate quantification - hot-start capability reduces primer-dimer formation and saves time Sensitive - achieve excellent results from difficult and low

6 SensiFAST SYBR Kits Features and Benefits: Accurate quantification - hot-start capability reduces primer-dimer formation and saves time Sensitive - achieve excellent results from difficult and low copy targets Rapid - optimized proprietary enzyme and unique buffer chemistry for fast cycling Flexible - compatible with all standard and fast cycling instruments Reliable - tested to deliver highest reproducibility and reliability Product PACK SIZE Cat No. 200 Reactions BIO SensiFAST SYBR No-ROX Kit 500 Reactions BIO Reactions BIO Reactions BIO SensiFAST SYBR Hi-ROX Kit 500 Reactions BIO Reactions BIO Reactions BIO SensiFAST SYBR Lo-ROX Kit 500 Reactions BIO Reactions BIO Reactions BIO SensiFAST SYBR & Fluorescein Kit 500 Reactions BIO Reactions BIO Sensitivity The SensiFAST SYBR Kit has been developed for fast, highly sensitive and reproducible real-time PCR and has been validated on commonly used real-time instruments. The antibody-mediated hot-start DNA polymerase system reduces the chances of primer/dimers formation, allowing a much greater dynamic range by removing competition for reaction components during amplification, leading to greater sensitivity (fig. 1). The addition of the latest advances in buffer chemistry and enhancers also ensures that the SensiFAST SYBR Kits produce fast (under 30 minutes), highly-specific and reproducible (fig. 2) real-time PCR results. For ease-of-use and added convenience, SensiFAST SYBR Hi-ROX is provided as a 2x mastermix containing all the components necessary for real-time PCR, including the SYBR Green I dye, dntps, stabilisers and enhancers. The kits are suitable for DNA templates or RNA templates following reverse transcription with the Bioline Tetro cdna Synthesis Supermix. b) Melt curve Fig. 1 Comparison of SensiFAST SYBR & Fluorescein (red line) against another leading supplier Q (blue line) using fast cycling conditions. A fragment of ubiquitin gene was amplified using SensiFAST SYBR & Fluorescein (red) and the results were compared with amplifications using a Kit from supplier Q (blue). The process used a 10 fold serial dilution of human RNA (in triplicate) over 5 orders of magnitude. a) The results exhibit that SensiFAST SYBR & Fluorescein was faster (earlier Ct) and more sensitive than supplier Q. b) There was far less primer-dimer formation with SensiFAST SYBR & Fluorescein. Specificity The SensiFAST SYBR Hi-ROX and Lo-ROX Kit contain premixed ROX and the SensiFAST SYBR & fluorescein contain premixed fluorescein, for optional use as a passive reference. Fig. 2 Comparison of SensiFAST SYBR Hi-ROX (blue line) against another leading supplier (red line) using fast cycling conditions. A fragment of ubiquitin gene was amplified using SensiFAST SYBR Hi-ROX (blue) and the results were compared with amplifications using a Kit from supplier Q (red). The process used a 10 fold serial dilution of human cdna (in quadruplicate) over 4 orders of magnitude. The results illustrate SensiFAST SYBR Hi-ROX was faster (earlier Ct). At low concentration supplier Q has a lower yield of product. 6 For more information please visit

Superior Fast DNA Analysis SensiFAST Probe Kits Features and Benefits: Highly specific - minimal non-specific activity leading to better efficiency and greater accuracy Efficient multiplexing -

7 Superior Fast DNA Analysis SensiFAST Probe Kits Features and Benefits: Highly specific - minimal non-specific activity leading to better efficiency and greater accuracy Efficient multiplexing - increasing throughput, reducing reaction cost and conserving precious sample material Fast - optimized for fast cycling conditions, ideal for high-throughput Ultra-sensitive - perfect for difficult and low copy number samples Reliable - tested to deliver highest reproducibility and reliability Product PACK SIZE Cat No. 200 Reactions BIO SensiFAST Probe Hi-ROX Kit 500 Reactions BIO Reactions BIO Reactions BIO SensiFAST Probe Lo-ROX Kit 500 Reactions BIO Reactions BIO Reactions BIO SensiFAST Probe No-ROX Kit 500 Reactions BIO Reactions BIO Sensitivity and reproducibility The SensiFAST Probe Kit has been developed for fast, highly reproducible real-time PCR and has been validated on all commonly used real-time instruments. The kit has been formulated for use with probe-detection technology, including TaqMan, Scorpions and molecular beacon probes. SensiFAST Probe Kit has been optimized for fast mode on fast qpcr instruments and fast cycling conditions on standard qpcr instruments. A combination of the latest advances in buffer chemistry and PCR enhancers, together with a hot-start DNA polymerase, ensures that the SensiFAST Probe Kit delivers shorter run times, is highly reproducible, highly-specific and ultrasensitive (fig.1), allowing more samples to be run with the highest confidence, in a day, ideal for high-throughput assays. The advanced buffer chemistry and enhancers also make SensiFAST Probe very sensitive and perfect for multiplexing (fig. 2). The kits are suitable for DNA templates or RNA templates following reverse transcription with the Bioline Tetro cdna Synthesis Supermix. Fig. 1 Comparison of sensitivity and reproducibility of SensiFAST Probe and Supplier I Kit. A fragment of GAPDH gene was amplified using SensiFAST Probe (Red) and the results were compared with amplifications using a Kit from supplier I (Green). The process used a 10 fold serial dilution of human DNA (in quadruplicate) over several orders of magnitude. The results illustrate that SensiFAST Probe is far more sensitive than supplier I. Sensitivity and efficiency in multiplexing Efficiencies b) Singleplex standard curve Multiplex standard curve The SensiFAST Probe Hi-ROX and Lo-ROX Kit contain premixed ROX for optional use. Fig. 2 Comparison of sensitivity and efficiency in a singleplex and quadruplex reaction A 10 fold serial dilution of human cdna was amplified with four different probes, both in singleplex reactions (blue line) and a quadruplex reaction (red line) (the results displayed are for the γ-actin and JOE dye). Five replicates were run using a conventional TaqMan primer/probe set under fast cycling conditions (3 min 95 C followed by 45 cycles 95 C 10s, 60 C 10s), on a Qiagen Rotor-Gene SensiFAST Probe No-ROX illustrates exactly the same high sensitivity, excellent reproducibility and Ct values for both the singleplex and multiplex reactions a) and no reduction of efficiency b) that is commonly associated with multiplexing. 7

8 SensiFAST HRM Kit Product PACK SIZE Cat No. Features and Benefits: 200 Reactions BIO Ultra-sensitive - accurately discriminates class 4 (A/T) SNP mutations SensiFAST HRM Kit 500 Reactions BIO Reactions BIO Highly reproducible - highly-specific, high- Sensitivity resolution melt analysis Rapid - enabling accurate results to be achieved faster Optimized - assays quickly and reliably established, even for difficult genomic loci Reliable performance - on any HRM capable thermo cycler High Resolution Melt (HRM) curve analysis is a post-pcr analysis method of characterizing nucleic acid samples based on DNA strand dissociation behaviour during transition from double-stranded DNA to single-stranded b) DNA with increasing temperature. It uses intercalating dyes, highly accurate melt curves and the application of specific statistical analyses. SensiFAST HRM has been developed using the latest Normalised minus history 31/1/2011 A advances in buffer chemistry and enhancers, together with an antibody-mediated hot-start DNA polymerase system, to ensure that the SensiFAST HRM Kit delivers fast, reproducible, highly-specific and ultra-sensitive c) deg. HRM analysis, ideal for small and medium sized genotyping projects. No. Hue Name Genotype Confidence % 25 SF HRM Monoduplex A A SF HRM Monoduplex A A SF HRM Monoduplex A A The combination of our SensiFAST DNA Polymerase, buffer and EvaGreen dye enables amplification and 28 SF HRM Monoduplex A A SF HRM Monoduplex T T SF HRM Monoduplex T T SF HRM Monoduplex T T discrimination of even the most challenging sequence differences, such as class 4 SNPs (Fig. 1) without sequence preference. Main applications of HRM include Single 32 SF HRM Monoduplex T T SF HRM Hetroduplex AT AT SF HRM Hetroduplex AT AT SF HRM Hetroduplex AT AT SF HRM Hetroduplex AT AT Nucleotide Polymorphisms (SNPs) genotyping, epigenetics (DNA methylation analysis), zygosity testing (DNA mapping and DNA fingerprinting) and gene scanning (search for the presence of unknown variations in PCR amplicons). Fig. 1 Detection of an A/T (Class 4) SNP in a 84 bp amplicon a) High Resolution Melt Curve showing a sharp decrease in fluorescence when the double-stranded DNA melts into its single-stranded form. Green = homozygous with thymidine, Pink = homozygous with adenine, Black = heterozygous. b) Using SensiFAST HRM, the melt curves for the three alleles are clearly resolved, even with smaller amplicons, resulting in unambiguous discrimination between genotypes. c) Each sample was genotyped with over 98% confidence. The experiment was performed on a Qiagen Rotor-Gene 6000 instrument. 8 For more information please visit

9 Superior Fast Genotyping SensiFAST Genotyping Kit Features and Benefits: Accurate allele calling - clear allele discrimination for higher confidence genotype calling Tight fluorescence clusters - perfect for difficult SNPs Faster run time - enabling more experiments per day, ideal for large genotyping projects Highly reproducibility - increasing confidence in your results Designed for dual-labeled probe assays - for example TaqMan probe-based assays The SensiFAST Genotyping Kit has been developed for fast, precise and highly reproducible genotyping of sequence variants, including loci with type IV SNPs. The SensiFAST Genotyping Kit is a combination of the latest advances in buffer chemistry, together with an antibody-mediated hot-start DNA polymerase system. This ensures that the SensiFAST Genotyping Kit produces highly-specific, ultrasensitive real-time PCR with clear allelic discrimination and outstanding allele clustering (fig. 1). The SensiFAST Genotyping Kit is compatible with many dual-labeled probe assays (including TaqMan probe based assays) and has been validated on commonly used real-time instruments. The advanced buffer chemistry in the SensiFAST Genotyping Kit provides higher stringency and more specific binding of the allele-specific probes, resulting in much narrower probe melting temperature windows. The combination of these developments leads to wider and clearer separation of allele clusters (fig. 2) it allows robust genotype calling enabling reproducible and accurate results. The inclusion of an optimized protocol and the advanced buffer means that minimal optimization is required to achieve fast, reproducible and accurate results, ideal for high-throughput in large scale genotyping projects. Product PACK SIZE Cat No. SensiFAST Genotyping Kit Fluorescence ( ) 200 Reactions BIO Reactions BIO Reactions BIO Allelic clustering and discrimination a) SensiFAST Genotyping Kit A b) Supplier A A Endpoint Fluorescence Scatter Plot Fluorescence ( ) Fluorescence ( ) A/G Endpoint Fluorescence Scatter Plot A/G Fig. 1 DNA samples from ninety six Caucasian patients were genotyped using a custom TaqMan drug metabolism genotyping assay. This assay was used to detect an A/G SNP on gene CYP11B1 (a cytochrome P450 superfamily member). The SensiFAST Genotyping Kit a) was compared to a kit from Supplier A b) on the Roche LightCycler 480 (autocaller confidence level 95%). The results illustrate that the SensiFAST Genotyping Kit gives defined clusters whilst Supplier A gives trailing clusters and low confidence calling. G G Fluorescence ( ) 9

SensiFAST SYBR s Features and Benefits: Accurate quantification - hot-start capability reduces primer-dimer formation and saves time Sensitive - achieve excellent results from difficult and low copy

and fast cycling instruments The SensiFAST SYBR s has been developed for fast RT-qPCR and has been validated on all commonly used real-time instruments.

10 SensiFAST SYBR s Features and Benefits: Accurate quantification - hot-start capability reduces primer-dimer formation and saves time Sensitive - achieve excellent results from difficult and low copy targets Rapid - optimized proprietary enzyme and unique buffer chemistry for fast cycling Reliable - tested to deliver highest reproducibility and reliability Flexible - compatible with all standard and fast cycling instruments The SensiFAST SYBR s has been developed for fast RT-qPCR and has been validated on all commonly used real-time instruments. The SensiFAST SYBR One- Step Kits has been formulated for highly reproducible first-strand cdna synthesis and subsequent real-time PCR in a single tube. The antibody-mediated hot-start DNA polymerase system reduces the chances of primer/ dimers formation, allowing a much greater dynamic range by removing competition for reaction components during amplification, leading to greater reproducibility (fig. 1). The addition of the latest advances in buffer chemistry and enhancers also ensures that the SensiFAST SYBR One- Step Kits produce fast, highly-specific and sensitivity onestep RT-qPCR (fig. 2). Product PACK SIZE Cat No. SensiFAST SYBR No-ROX SensiFAST SYBR Hi-ROX SensiFAST SYBR Lo-ROX SensiFAST SYBR & Fluorescein Fluorescence ( ) Fluorescence ( ) Reproducibility 1.0E1 1.0E Cycles 100 Reactions BIO Reactions BIO Reactions BIO Reactions BIO Reactions BIO Reactions BIO Reactions BIO Reactions BIO b) Melt curve Fig. 1 Comparison of SensiFAST SYBR Lo-ROX One-Step (red line) against another leading supplier (green line) using fast cycling conditions. A fragment of the human ß-actin gene was amplified using SensiFAST SYBR Lo-ROX One-Step (red) and the results were compared with amplifications using s from supplier A (green). The process used a 10 fold serial dilution of human RNA (in triplicate) over 5 orders of magnitude. a) The results illustrate that the SensiFAST SYBR Lo-ROX One-Step Kit was faster (earlier Ct) and much more reproducible than b) With less primer dimers. Sensitivity and Specificity The SensiFAST s consist of a 2x SensiFAST SYBR One-Step mix, plus separate reverse transcriptase and RiboSafe RNase Inhibitor. SensiFAST SYBR One- Step Hi-ROX and Lo-ROX Kit contain premixed ROX and the SensiFAST SYBR & fluorescein contain premixed fluorescein for optional use as a passive reference. b) Melt curve 10 For more information please visit Fig. 2 Comparison of SensiFAST SYBR Hi-ROX One-Step (red line) against another leading supplier (green line) using fast cycling conditions. A fragment of ubiquitin gene was amplified using SensiFAST SYBR Hi-ROX One-Step (red) and the results were compared with amplifications using a Kit from supplier I (green). The process used a 10 fold serial dilution of human RNA over 5 orders of magnitude. The results illustrates that SensiFAST SYBR Hi-ROX One-Step was faster (earlier Ct) and more sensitive than supplier I. a) SensiFAST SYBR Hi-ROX One-Step was faster (earlier Ct) and more sensitive than supplier I. b) Primer-dimer is seen in the supplier I but not in SensiFAST.

Superior Fast RNA Analysis SensiFAST Probe s Features and Benefits: Accurate quantification - for calculating relative gene expression from RNA samples Sensitive - unique buffer chemistry gives

deliver highest reproducibility and reliability The SensiFAST Probe has been optimized for fast mode on fast RT-qPCR instruments and fast cycling conditions on standard qpcr instruments.

11 Superior Fast RNA Analysis SensiFAST Probe s Features and Benefits: Accurate quantification - for calculating relative gene expression from RNA samples Sensitive - unique buffer chemistry gives superior sensitivity of low copy targets Fast - optimized proprietary enzyme and unique buffer chemistry for fast cycling Flexible - compatible with all fast cycling instruments Reliable - tested to deliver highest reproducibility and reliability The SensiFAST Probe has been optimized for fast mode on fast RT-qPCR instruments and fast cycling conditions on standard qpcr instruments. The kit is designed for superior sensitivity and specificity with probe-detection technology, including TaqMan, Scorpions and molecular beacon probes. The SensiFAST Probe s have been formulated for highly reproducible first-strand cdna synthesis and subsequent real-time PCR in a single tube. The antibody-mediated hot-start DNA polymerase system reduces the chances of primer/dimers formation, allowing a much greater dynamic range by removing competition for reaction components during amplification, leading to greater sensitivity (fig. 1). The advanced buffer chemistry and enhancers also gives SensiFAST Probe One-Step unbeatable efficiency in multiplexing (fig. 2). The SensiFAST Probe s consists of a 2x SensiFAST Probe One-Step mix, separate reverse transcriptase and RiboSafe RNase Inhibitor. SensiFAST Probe Hi-ROX and Lo-ROX s also contain premixed ROX for optional use. Fluorescence ( ) Fluorescence ( ) Product PACK SIZE Cat No. SensiFAST Probe Hi-ROX SensiFAST Probe Lo-ROX SensiFAST Probe No-ROX Specificity Cycle Number Cycles 100 Reactions BIO Reactions BIO Reactions BIO Reactions BIO Reactions BIO Reactions BIO b) Standard curve Log Concentration Fig. 1. Comparison of SensiFAST Probe One-Step (red line) against a leading supplier using fast cycling conditions. A fragment of Mouse ß-actin amplified in triplicate using gene specific primers and TaqMan probe according to each manufacturer s protocol, from 10-fold serial dilution of RNA with either SensiFAST Probe One-Step (red) and competitor mix A (green). a) The results illustrate that SensiFAST Probe is faster by four Cts and more sensitive than supplier A. (more than 10 fold). b) The standard curve shows an efficiency of 98% together with excellent specificity. Sensitivity and reproducibility Cycles Fig. 2. Sensitivity and reproducibility of SensiFAST Probe One-Step. A fragment of the mouse b-actin gene was amplified from a 10-fold serial dilution of total RNA from mouse 3T3 cells in triplicate using SensiFAST Probe, primers and a TaqMan probe, using fast cycling conditions (40 cycles 95 C 10s, 60 C 30s). The results illustrate that that the SensiFAST Probe works reproducibly and efficiently with fast protocols

www.bioline.com United Kingdom Bioline Reagents Ltd 16 The Edge Business Centre Humber Road London NW2 6EW Tel: +44 (0)20 8830 5300 Fax: +44 (0)20 8452 2822 email: info.uk@bioline.

12 United Kingdom Bioline Reagents Ltd 16 The Edge Business Centre Humber Road London NW2 6EW Tel: +44 (0) Fax: +44 (0) Germany Bioline GmbH Im Biotechnologiepark, TGZ 2 D Luckenwalde Tel: +49 (0) Fax: +49 (0) info.de@bioline.com Australia Bioline (Aust) Pty Ltd PO Box 122 Alexandria NSW 1435 Tel: +61 (0) Fax: +61 (0) info.aust@bioline.com USA Bioline USA Inc. 305 Constitution Drive Taunton MA Tel: Fax: Order Toll Free: info.us@bioline.com To download the electronic version of this guide, please go to PG0413V2.0

Exceed the limit. SensiFAST Real-Time PCR Family. Superior Sensitive Quantification

Exceed the limit. SensiFAST Real-Time PCR Family. Superior Sensitive Quantification Exceed the limit SensiFAST Real-Time PCR Family Superior Sensitive Quantification SensiFAST Real-Time PCR Kits Fast - optimized for fast cycling conditions (under 30 minutes), ideal for high-throughput