Amplification: ConsumablesAmplification Reagents and Plastics
Factors Impacting Gene Expression AnalysisRNA Isolation RNA integrity, purity, and yield■■ Genomic DNA contamination■■ Inhibitors of cDNA synthesis and qPCR■■ RNase and DNase contamination■■Reagents — Reverse Transcription cDNA synthesis efficiency■■ RNA protection■■ Input RNA capacity■■ Accurate representation of mRNA■■Reagents — Real-Time qPCR Detection sensitivity■■■■ Assay specificity■■ Inhibitors in sample■■ Reproducibility of thermal cycling conditions and instrument compatibilityPCR Plastic Consumables Instrument compatibility■■ Optimum performance■■ Automation friendly■■ Potential source of contamination and inhibition■■
RNA Isolation■■ Kits are designed and formulated to assist in the isolation of highly pure and intact RNA from different starting materials■■ RNA is compatible with a variety of downstream applications – Real-time qPCR – Northern blotting – Microarray analysis – cDNA library construction■■ DNase treatment ensures genomic DNA removal
RNA Isolation iScript™ RT-qPCR Sample Preparation Reagent PureZOL™ RNA Isolation Reagent ■■ Reagent stabilizes RNA and removes genomic DNA in less than 10 min ■■ Single-solution format permits recovery of RNA from small quantities of ■■ Suitable for adherent or suspension animal cells tissues or cells, making it ideally suited for gene expression studieswww.bio-rad.com/iscript ■■ RT-qPCR is directly enabled from cells without RNA purification when ■■ Efficient RNA purification from cultured cells, yeast, viruses, and bacteria, combined with an iScript reverse transcription kit and real-time supermix as well as plant and animal tissues ■■ Reagent allows multiplex real-time detection of up to 4 targets from as few ■■ PureZOL efficiently lyses cells and tissues, deproteinates RNA, and as 10 cells inactivates endogenous nucleases in a single step ■■ Ideal for rapid, high-throughput gene expression analysis ■■ Scalable starting sample amount For more information, request bulletin 5736. ■■ Convenient isolation of RNA, DNA, and protein from the same sample 104 Aurum™ Vacuum Manifold ■■ Vacuum-mediated nucleic acid purification platform ■■ Versatile manifold format adaptable for 96-well plate or up to 10 3 18 spin columns RFU ■■ Manifold ensures fast, high-quality sample preparation while maintaining the simplicity of vacuum processing 10 2 ■■ Unique vacuum regulator design allows for complete control of negative pressure 0 10 20 30 40 Cycles iScript RT-qPCR sample preparation reagent generates linear results over varying input cell amounts. HeLa cells (125, 25, 5, and 1 cells/µl) were treated and analyzed for GAPDH expression levels using iScript cDNA synthesis kit and iQ ™ SYBR ® Green supermix on the CFX96™ real-time PCR detection system. RFU, relative fluorescence units. 732-6890, 100 ml PureZOL RNA Isolation Reagent 170-8899, 5 x 10 ml 732-6470, 1 unit iScript RT-qPCR Sample Preparation Reagent Aurum Vacuum Manifold Amplification Reagents and Plastics 6 Visit us on the Web at www.bio-rad.com.
Reagents —Reverse Transcription■■ Formulated for efficient reverse transcription across a broad linear dynamic range■■ Potent RNase A inhibitors protect RNA during setup and reverse transcription■■ Flexible input RNA capacity to suit different experimental needs■■ Optimized for gene expression analysis using real-time qPCR
Reagents Two-Step Reverse Transcription Reagents iScript™ Advanced cDNA Synthesis Kit for RT-qPCR iScript Reverse Transcription Supermix ■■Increased qPCR data throughput and cost effectiveness from a single 20 µl for RT-qPCRwww.bio-rad.com/ reverse transcription (RT) reaction ■■ 1-tube format for simple and fast setup, and reduced pipetting variabilityiscript ■■ Superior sensitivity and broad linear dynamic range for RT (7.5 µg–100 fg) ■■ Liquid format at –20°C offers superior stability and eliminates freeze/thaw cycle ■■ 2-tube kit (5x iScript reaction mix and iScript reverse transcriptase) for ease ■■ Superior sensitivity and broad linear dynamic range for RT (1 µg–100 fg) of use and reduced reaction setup time ■■ Optimized blend of oligo(dT) and random primers ensures complete and ■■ Optimized blend of oligo(dT) and random primers ensures complete and unbiased RNA sequence representation unbiased RNA sequence representation ■■ RNase H+ MMLV reverse transcriptase (preblended with RNase inhibitor) ■■ RNase H+ MMLV reverse transcriptase (preblended with RNase inhibitor) delivers high sensitivity for real-time RT-qPCR and eliminates additional delivers high sensitivity for real-time RT-qPCR and eliminates additional RNase H+ step RNase H+ step ■■ Potent blend of RNaseA inhibitor protects RNA during setup and RT ■■ Potent blend of RNaseA inhibitor protects RNA during setup and RT ■■ Short 40 min protocol allows fast qPCR data generation ■■ Short 35 min protocol allows fast qPCR data generation For more information, request bulletin 6031. For more information, request bulletin 6125. 35 104 — 1 µg RNA Average Cq SD CV, % — 100 ng 103 104 100 ng 21.35 0.123 0.576 30 — 10 ng RFU — 1 ng 100 pg 31.56 0.147 0.465 102 — 100 pg 25 — 10 pg RFU RFU — 1 pg Cq 0 10 20 Cycles 30 40 103 20 103 102 15 –2 –1 0 1 2 3 4 0 10 20 30 40 10 20 30 40 log starting quantity Cycles Cycles iScript advanced cDNA synthesis kit for RT-qPCR provides iScript reverse transcription supermix for RT-qPCR efficiently Excellent data reproducibility. PGK-1 mRNA (~160 bp), a gene that superior sensitivity and a broad linear dynamic range for reverse reverse transcribes RNA over a broad linear dynamic range encodes a glycolytic enzyme, was quantified using iScript reverse transcription. Total RNA (7.5 µg–1 pg) from HeLa cells was reverse for reliable gene expression analysis data. Different amounts of transcription supermix for RT-qPCR both with 100 ng () and 100 pg () transcribed using the iScript advanced cDNA synthesis kit for HeLa cell RNA (amounts shown in inset) were reverse transcribed of input RNA. For each input RNA, 48 individual RT reactions were RT-qPCR in a 20 µl reaction. A tenfold dilution of generated cDNA and one-tenth of the resulting cDNA was used as a template to performed and one-tenth of the resulting cDNA was used in the qPCR was used as template to amplify α-tubulin in a 10 µl qPCR reaction amplify β-actin gene (~90 bp) in 20 µl qPCR reactions with iQ™ reaction with SsoFast™ probes supermix. The gene expression analysis with iQ™ SYBR® Green supermix on a CFX384™ real-time PCR SYBR® Green supermix. Standard curve R 2 = 0.999, efficiency = data show excellent reproducibility both with high and low levels of input detection system. Efficiency = 90.7%, R2 = 0.999, slope = –3.57. Cq, 99.7%, slope = –3.33. RFU, relative fluorescence units. target mRNA. The ~10 Cq difference for the 1,000-fold dilution of RNA quantification cycle; RFU, relative fluorescence units. (100 ng–100 pg) demonstrates good reverse transcription efficiencies across different input RNAs. Cq, quantification cycle; RFU, relative fluorescence units. Amplification Reagents and Plastics 10 Visit us on the Web at www.bio-rad.com.
Reagents One-Step RT-qPCR Reagents iScript™ One-Step RT-PCR Kit with SYBR® Green Benefits of iScript one-step kits: ■■ For use on a broad range of real-time PCR instruments ■■ Provide powerful combination of iScript RNase H+ reverse transcriptasewww.bio-rad.com/iscript ■■ Extremely sensitive detection (100 ng–1 pg) of input RNA and antibody-mediated hot-start iTaq™ DNA polymerase Are ideal for rapid, high-throughput gene expression analysis iScript One-Step RT-PCR Kit for Probes ■■ ■■ For use with all types of hybridization probes, including dual-labeled ■■ Perform cDNA synthesis and qPCR in 1 tube, minimizing handling and oligonucleotide probes, FRET probes, and molecular beacons contamination risk ■■ Extremely sensitive detection (1 µg–1 pg) of input RNA For more information, request bulletin 3066. PCR baseline-subtracted curve fit, RFU PCR baseline-subtracted curve fit, RFU 10,000 1,000 1,000 100 100 10 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 Cycles Cycles iScript™ one-step RT-PCR kit with SYBR® Green provides high reproducibility and iScript one-step RT-PCR kit for probes delivers unparalleled results over an sensitivity across a broad range of concentrations. Reactions were performed in triplicate, extremely wide dynamic range. RNA (1 µg–100 fg) isolated from HeLa cells using along with no-template controls, using GAPDH primers and 100 ng–100 fg total HeLa RNA. the Aurum™ total RNA kit was reverse transcribed and amplified using primers to Reactions were carried out on the iCycler iQ® real-time detection system. Standard curve β-actin and a FAM-labeled detection probe. Each dilution was performed in triplicate r = 1.000, efficiency = 95%, slope = –3.47. RFU, relative fluorescence units. and RT-PCR was carried out on the iCycler iQ detection system. Standard curve r = 1.000, efficiency = 97.2%, slope = –3.39. RFU, relative fluorescence units. Ordering Information Catalog # Description $ Two-Step Reverse Transcription Reagents One-Step RT-qPCR Reagents 170-8842 iScript Advanced cDNA Synthesis Kit for RT-qPCR, 50 x 20 μl reactions 170-8892 iScript One-Step RT-PCR Kit with SYBR Green, 50 x 50 μl reactions 170-8843 iScript Advanced cDNA Synthesis Kit for RT-qPCR, 250 x 20 μl reactions 170-8893 iScript One-Step RT-PCR Kit with SYBR Green, 200 x 50 μl reactions 170-8890 iScript cDNA Synthesis Kit, 25 x 20 μl reactions 170-8894 iScript One-Step RT-PCR Kit for Probes, 50 x 50 μl reactions 170-8891 iScript cDNA Synthesis Kit, 100 x 20 μl reactions 170-8895 iScript One-Step RT-PCR Kit for Probes, 200 x 50 μl reactions 170-8840 iScript Reverse Transcription Supermix for RT-qPCR, 25 x 20 μl reactions 170-8841 iScript Reverse Transcription Supermix for RT-qPCR, 100 x 20 μl reactions 170-8896 iScript Select cDNA Synthesis Kit, 25 x 20 μl reactions 170-8897 iScript Select cDNA Synthesis Kit, 100 x 20 μl reactions Amplification Reagents and Plastics 12 Visit us on the Web at www.bio-rad.com.
Reagents —Real-Time qPCR■■ Patented Sso7d fusion enzyme technology delivers higher processivity and inhibitor tolerance■■ Antibody-mediated hot-start polymerases enable instant activation and higher specificity■■ Choice of fast, standard, or universal cycling conditions■■ Formulated for optimal performance on a variety of real-time instruments Reagents
Reagents Real-Time qPCR Reagents Selection Guide SYBR® Green / EvaGreen Supermixes Probes Supermixes One-Step Kits for RT-qPCR SsoAdvanced™ SsoFast™ iQ™ SsoFast iTaq™ Fast iTaq™ EpiQ™ SsoFast iQ iQ SsoFast iTaq Fast iTaq iScript™ iScript SYBR® EvaGreen® SYBR® EvaGreen SYBR® Green SYBR® Chromatin Probes Supermix Multiplex Probes Supermix Supermix One-Step One-Step Green Supermix Green Supermix Supermix with Green SYBR® Supermix Powermix Supermix with ROX with ROX RT-PCR Kit RT-PCR Kit Supermix Supermix with Low ROX Supermix Green with ROX with SYBR® for ProbesReal-Time PCR Instrument ROX with ROX Supermix GreenBio-RadCFX96™, CFX96 Touch™,CFX384™, CFX384 Touch™, CFX ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔Connect™iQ™, iQ™5, MyiQ™, MyiQ™2 ✔ ✔ ✔ ✔ ✔ ✔■ ✔ ✔ ✔MiniOpticon™, DNA EngineOpticon® I and II ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔Applied BiosystemsStepOne/StepOne Plus ◆ ◆ ◆ ◆ ◆ ✔ ✔ ◆ ◆ ◆ ✔ ◆ ✔ ◆ ◆7500, ViiA7 ✔ ✔ ✔ ✔7000, 7300, 7700, 7900HT ✔ ✔ ✔StratageneMx3000P, 3005P, 4000 ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔EppendorfMastercycler eprealplex 2 or 4 ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔QIAGEN/CorbettRotor-Gene 3000, 6000, Q ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔RocheLightCycler 480 ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔LightCycler 1.0, 1.5, 2.0 ▲ ▲ ▲ ▲ ▲ ▲ ✔ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲Idaho TechnologyLightScanner HR-1 ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔LightScanner 32 ▲ ▲ ▲ ▲ ▲ ▲ ✔ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲✔ Recommended for use as is ◆ ROX reference setting must be turned “off” ▲ BSA must be added according to instrument specifications Amplification Reagents and Plastics 14 Visit us on the Web at www.bio-rad.com.
Reagents Real-Time qPCR — SYBR® Green/EvaGreen SsoFast™ EvaGreen® Supermix SsoFast EvaGreen Supermix with Low ROX ■■ Inhibitor tolerance and enhanced processivity with Sso7d fusion polymerase ■■ Blended with ROX for optimal performance on Applied Biosystemswww.bio-rad.com/ standard and fast 7500 real-time PCR instruments ■■ Robust formulation ensures maximum efficiency, sensitivity,supermixes and reproducibility for qPCR For more information, request bulletin 5919. ■■ Instant polymerase activation and rapid polymerization kinetics deliver fast qPCR results For more information, request bulletin 5816. 104 100.000 6,000 900 800 5,000 700 10.000 600 500 RFU 4,000 400 103 RFU RFU ∆Rn 300 3,000 1.000 200 100 2,000 0 0 10 20 30 40 0.100 Cycles 1,000 102 0 0.010 0 10 20 30 40 0 10 20 30 40 4 8 12 16 20 24 28 32 36 40 Cycles Cycles Cycles The unique fusion polymerase in SsoFast EvaGreen supermix SsoFast EvaGreen supermix demonstrates superior inhibitor The unique fusion polymerase in SsoFast EvaGreen supermix delivers extreme speed and generates exceptional qPCR results tolerance and direct qPCR capability from cell culture. Results with low ROX generates exceptional qPCR results on the ABI in less than 30 min. Tenfold serial dilutions of 10 ng–100 ag cDNA show efficient amplification and detection of a spike-in control 7500 fast real-time PCR system. Tenfold serial dilutions of 10 ng–100 fg from human spleen were used in each 20 μl reaction to detect template in the absence (■) or presence (■) of conditioned tissue 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 culture medium using SsoFast EvaGreen supermix. Inset shows a α-tubulin. α-tubulin efficiency = 105.8%, r = 0.996. Total qPCR run time = 29 min. RFU, relative fluorescence units. competitor’s standard qPCR reagent is able to amplify the spike-in time = 39 min (not including melt curve). control template only in the absence (■) vs. presence (■) of culture medium. RFU, relative fluorescence units. Amplification Reagents and Plastics 16 Visit us on the Web at www.bio-rad.com.
Reagents Real-Time qPCR — SYBR® Green/EvaGreen Precision Melt Supermix EpiQ™ Chromatin SYBR® Green Supermix ■■ Optimized formulation containing EvaGreen dye delivers robust PCR and ■■ Robust formulation delivers superior sensitivity and efficiency for qPCR fromwww.bio-rad.com/ high resolution melt (HRM) performance genomic DNA templatessupermixes ■■ Sensitive and effective discrimination of all 4 SNP classes across a broad ■■ Protocol is optimized for difficult real-time qPCR reactions for high GC amplicons range of amplicons ■■ Supermix contains fluorescein and ROX and is compatible with all real- ■■ Accurate detection of CpG methylation status for epigenetic studies time PCR instruments except Applied Biosystems 7000, 7300, 7700, and 7900 models (additional ROX can be added by ordering the dye ■■ Exceptional room temperature stability for high-throughput HRM studies separately, catalog #172-5858) ■■ Reliable performance on any HRM-capable thermal cycler Please refer to page 22 for more details about the EpiQ chromatin analysis kit. For more information, request bulletin 6137. For more information, request bulletin 6020. A B 0.20 0.02 0.0 –0.1 0.15 0.00 Difference RFU –0.2 Difference RFU Difference RFU 0.10 –0.02 –0.3 –0.04 0.05 –0.4 –0.06 –0.5 0.00 –0.08 –0.6 –0.05 78 79 80 81 82 83 75 76 77 78 79 80 74 76 78 80 82 84 Temperature, °C Temperature, °C Temperature, °C Precision melt supermix delivers robust HRM for single nucleotide polymorphisms (SNPs). Discrimination of class I Accurate methylation detection with precision melt supermix. Mixtures and IV SNP genotypes are shown in panels A and B, respectively. Class I (A to G substitution) and class IV (A to T substitution) SNP of methylated and unmethylated human genomic DNA of varying ratios were genotypes from mouse genomic DNA were analyzed using precision melt supermix. Wild type (■), heterozygote (■), and homozygous analyzed using HRM on a CFX384 real-time PCR detection system. Increasing mutant (■) are shown in the difference plots normalized to wild-type samples. HRM analysis was performed on a CFX384™ real-time PCR amounts of methylated DNA (■, 0%; ■, 2%; ■, 5%; ■, 50%; ■, 75%; ■, 95%; detection system and genotypes were automatically assigned by Precision Melt Analysis™ software. Amplification was carried out for 35 and ■, 100%) were analyzed for methylation of the human RARB2 gene. The cycles. Total run time including melt curve = 150 min. RFU, relative fluorescence units. genomic region contains 7 CpG sites and is 88 base pairs in length. Total run time including melt curve = 190 min. RFU, relative fluorescence units. Amplification Reagents and Plastics 18 Visit us on the Web at www.bio-rad.com.
Reagents Real-Time qPCR Probes iQ™ Multiplex Powermix iTaq™ Fast Supermix with ROX ■■ Robust supermix formulated for sensitive and efficient multiplex qPCR ■■ Developed and validated for Applied Biosystems 7500 standard and fastwww.bio-rad.com/ and Stratagene real-time PCR instrumentssupermixes ■■ Reliable quantification of up to 4 targets (expression levels can vary up to 106-fold between target genes) or up to 5 targets ■■ Compatible with standard and fast thermal cycling conditions ■■ Linearity over 6 orders of magnitude of input cDNA and 4 orders of ■■ Antibody-mediated iTaq DNA polymerase provided in a convenient magnitude of input genomic DNA 2x supermix ■■ Suitable for a wide variety of applications, including gene expression For more information, request bulletin 5737. analysis, SNP genotyping, SNP analysis, GMO detection, and viral load detection iTaq Supermix with ROX ■■ Developed and validated for Applied Biosystems 7000, 7300, 7700, 7900, For more information, request bulletin 5348. StepOne, and StepOne Plus real-time PCR instruments ■■ Sensitive and accurate detection over 6 orders of magnitude ■■ Hot-start iTaq DNA polymerase in optimized buffer for simplex and duplex qPCR For more information, request bulletin 3065. 10.000 PCR baseline-subtracted curve fit, RFU 1,000 1.000 ∆Rn ∆Rn 1.000 100 0.100 0.100 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 35 40 45 0 10 20 30 40 Cycles Cycles Cycles iQ multiplex powermix produces highly reliable qPCR results Robust duplex qPCR results with iTaq fast supermix with ROX on the iTaq supermix with ROX produces superior results on the ABI 7900HT for up to five targets in a single tube, with no difference in Applied Biosystems 7500 fast real-time PCR system. cDNA inputs: system. Total RNA from HeLa cells was reverse transcribed using the detection of a low-expressing gene in multiplex or singleplex. 100-fold serial dilutions of cDNA from the equivalent of 1 µg–1 pg total RNA iScript™ reverse transcription supermix for RT-qPCR. A tenfold dilution of One-tenth of a 1 µg cDNA synthesis reaction of human thymus total were used in each 20 µl reaction. FAM-labeled 18S rRNA probe duplex generated cDNA (100 ng–1 pg) was used as template to amplify the β-actin RNA was used in each 20 µl reaction. FAM-labeled β-actin probe (), reaction (), VIC-labeled beta-2-microglobulin (B2M) probe duplex gene with a FAM-labeled probe. Standard curve R2 = 0.999, efficiency = Cy5-labeled α-tubulin probe (), HEX-labeled GAPDH probe (), reaction (), VIC-labeled B2M probe singleplex reaction (). 18S rRNA 98.0%, slope = –3.38. TAMRA-labeled cyclophilin probe (), Texas Red–labeled IL-2 probe (). efficiency = 98.6%, r = 0.999; B2M efficiency = 98.0%, r = 0.998. Total RFU, relative fluorescence units. qPCR run time = 45 min. Amplification Reagents and Plastics 20 Visit us on the Web at www.bio-rad.com.