Reliance One-Step Multiplex RT-qPCR Supermix

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Reliance One-Step Multiplex RT-qPCR  Supermix | Bio-Rad.com

Overview

Exceptional Performance of 5-Target Multiplex

Five targets across seven orders of magnitude (1 pg to 1 μg RNA input) were amplified using Reliance One-Step Multiplex Supermix on a CFX96 Touch Real-Time System. The data demonstrated exceptional performance with superior efficiency and sensitivity over a wide dynamic range down to 1 pg of RNA input.

Exceptional 5-Plex Performance

Unparalleled Stability of Assembled Reactions

Five targets across seven orders of magnitude (1 pg to 1 μg RNA input) were amplified using various reagents on a CFX96 Touch Real-Time System immediately after the reaction set up and then again after 24 hours incubation at room temperature protected from light. Reliance One-Step Multiplex Supermix maintained less than 1 ΔCq between two runs () indicating uncompromised performance even after 24 hour incubation of assembled reactions while other suppliers’ reagents showed significantly declined performance with over 1 ΔCq () or no amplification ().

Exceptional Room Temperature Stability

Enhanced Tolerance to Inhibitors

Comparison of Inhibitor Tolerance of Reliance One-Step Multiplex Supermix and Products from Other Suppliers

Comparison of Inhibitor Tolerance of Reliance One-Step Multiplex Supermix and Products from Other Suppliers

Two inhibitors of RT-PCR — ethanol and hematin, were spiked in to 5-plex one-step RT-PCR reactions prepared using three one-step RT-PCR reagents to assess the magnitude of Cq shift caused by the inhibitors. The Reliance One-Step Multiplex Supermix demonstrated robust performance against products from other suppliers with minimal dCq in reactions containing ethanol or hematin.

dCq = Ave. Cq 5-plex reactions with inhibitor — Ave. Cq 5-plex reactions without inhibitor



Enhanced Tolerance to Ethanol

Enhanced tolerance to ethanol

5-plex reactions were prepared using Reliance One-Step Multiplex Supermix with two RNA inputs of 10 pg and 200 pg. Ethanol was added to the reactions at a final concentration of 0%, 0.25%, 1.25, and 2.5 to assess the magnitude of Cq shift caused by varying ethanol volumes. The Reliance One-Step Multiplex Supermix maintained less than 1 dCq across all reactions up to 1.25% indicating excellent tolerance to ethanol. It also demonstrated minimal inhibition even at a high concentration of 2.5%.

Co-detection of RNA and DNA Targets in 5-Plex Reaction

An experiment was designed to test samples containing three viral nucleic acids, two in their RNA form — Zika (ZIKV) and Chikungunya (CHIKV) — and the DNA version of Dengue Virus (DENV), as well as an internal positive control RNA and human genomic DNA control (gDNA). The viral targets were tested at five different dilutions, while the controls were kept constant. The number of copies for the viral RNA targets was determined by Droplet Digital RT-PCR and was diluted to a single copy or less per reaction in their lowest dilution series (Table 1). Two identical plates each containing 10 technical replicates of each sample were prepared; one plate was analyzed immediately (time = 0 hr) while the second plate was stored at room temperature (25 °C) and analyzed the next day (time = 24 hr). Reliance One-Step Multiplex Supermix efficiently detected both RNA and DNA targets with outstanding efficiency and single copy detection sensitivity at both time points (Fig 1.)

Table 1. Targets and templates used for a 5-plex reaction.


Target Reporter Dye Template Amount RNA Copies
ZIKV FAM Viral RNA 5 dilution series 1.17 to 11,668
CHIKV HEX Viral RNA 5 dilution series 0.15 to 1,498
DENV Texas Red Synthetic DNA 5 dilution series N/A
IPC Cy5 Synthetic DNA Constant N/A
RNaseP Cy5.5 Human gDNA Constant N/A

Co-detection of DNA and RNA Targets in 5-plex Reaction
Figure 1. Co-detection of various nucleic acid targets in 5-plex reaction.

Exclusive Sso7d Fusion Polymerase Technology to Empower Your PCR

Sso7d, a 7 kD protein obtained from Sulfolobus solfataricus, binds to dsDNA without any sequence specificity.

Sso7d is covalently linked to a DNA polymerase and is able to stabilize the polymerase-template complex without compromising the structural integrity, thermal stability, or catalytic activity of the enzyme.

The unique structure of this Sso7d fusion polymerase provides several significant advantages over ordinary DNA polymerase:

  • Increased processivity
  • Effective amplification in GC-rich regions or strong secondary structure
  • Efficient amplification of longer products
  • Superior tolerance to PCR inhibitors
Sso7D Technology
Use On Any qPCR System

Passive reference dye signal in low- and high-ROX instruments and using Bio-Rad’s universal reagents.

A. Optimal excitation of the ROX dye at ~575 nm in low-ROX instruments results in a strong ROX signal, while poor excitation at ~488 nm in high-ROX instruments leads to a weak ROX signal, creating a need for additional ROX when using high-ROX instruments. B. A blend of dyes in Bio-Rad’s universal reagents allows excitation of the ROX dye in low-ROX instruments and the long Stoke-shift dye in high-ROX instruments, negating the need for additional ROX in the latter case. Excitation (—); emission (– – –).

Universal Reference Dye Figure A

Universal Reference Dye Figure B

Reliance One-Step Multiplex Supermix is a ready-to-use, room-temperature stable, 4x reaction master mix optimized for sensitive amplification of up to 5 targets in a single reaction.
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Description

CFX Loyalty Program

Earn a free one-year service contract on your CFX System with purchase of Reagents.

CFX Loyalty Program

Use the Reliance One-Step Multiplex Supermix for sensitive amplification of up to 5 targets in a single reaction. Expertly formulated with high-performance enzymes and advanced reaction buffer, this robust supermix delivers reproducible RT-qPCR performance in a multiplex fashion, including with challenging templates, difficult targets or in the presence of PCR inhibitors. With its exceptional room temperature stability for assembled reactions, it can be easily integrated into an automated workflow. Reliance One-Step Multiplex Supermix is an ideal solution for multiplexed RT-qPCR, especially for pathogen/viral detection and high throughput screening applications.

Features and Benefits



Exceptional Performance of 5-Target Multiplex

Reliance One-Step Multiplex Supermix enables researchers to get the most data out of their samples. Optimally formulated with a novel proprietary Reliance Reverse Transcriptase and robust DNA polymerases blend, this supermix amplifies up to 5 targets across a broad linear dynamic range from 1 pg to 1 µg of RNA input with maximum sensitivity and efficiency.




Unparalleled Room Temperature Stability for Up To 24 Hours

Reliance One-Step Multiplex Supermix is developed with high-throughput researchers in mind. Whether you want to queue up plates ahead of time or use an automated workflow, Reliance One-Step Multiplex Supermix will maintain its performance even after 24 hour incubation of assembled reactions.




Efficient Co-detection of RNA and DNA Targets

Reliance One-Step Multiplex Supermix is designed to efficiently detect both RNA and DNA targets in the same reaction. Whether samples are tested for both RNA and DNA viruses at the same time or are run with internal controls, Reliance One-Step Multiplex Supermix reliably amplifies multiple targets regardless of their nature.

View Application Note (PDF 143KB)




Streamline Your Workflow

4x Reliance One-Step Multiplex Supermix is Formulated with:

  • Optimal blend of Reliance chimeric reverse transcriptase* and high-performance DNA polymerases including Sso7d fusion DNA polymerase**
  • 4X reaction buffer containing dNTPs, RNase inhibitor, and other reaction optimizing components
  • Reaction stabilizers
  • Patented*** universal reference dye

Applications and Uses

  • Gene expression analysis
  • Viral/pathogen detection (RNA viruses, DNA viruses, bacterial viruses, and fungal viruses)
  • Diagnostic research
  • High-throughput screening (small molecules, drug discovery, dosage experiment, CRISPR screening, and toxicology study)

Instrument Compatibility

Reliance One-Step Multiplex Supermix is compatible with Bio-Rad Real-Time PCR Detection Systems and commercially available systems listed below:

Systems
Applied Biosystems
  • StepOne/StepOnePlus
  • 7500, ViiA 7
  • 7000, 7300, 7700, 7900HT, QuantStudio Systems
Stratagene
  • Mx3000P, 3005P, 4000
Eppendorf
  • Mastercycler ep realplex 2, 4
QIAGEN
  • Corbett Rotor-Gene 3000, 6000, Q
Roche
  • LightCycler 480, 96
  • LightCycler 1.0, 1.5, 2.0

Choose the Right One-Step RT-qPCR Product for Your Needs

  Reliance One-Step Multiplex Supermix iTaq Universal One-Step Probe Kit
Key Features
  • Sensitive, efficient amplification of up to 5 targets
  • Room temperature stable up to 24 hours
  • Reliable amplification of up to 2 targets
  • Compatible with a fast protocol
Ideal for
  • Viral/pathogen detection in high-multiplex reaction
  • High throughput screening
  • Viral/pathogen detection
  • Singleplex and duplex reactions

Supporting Documents

  • Amplification Reagents and Plastics Brochure
  • SingleShot Cell Lysis Kit Instruction Manual
  • One-Step Multiplex RT-qPCR Using PrimePCR Probe Assays Protocol
  • Sensitive Codetection of Viral-Pathogen RNA and DNA Using One-Step Multiplex RT-qPCR Application Note

Online Resources

What is Real-Time PCR (qPCR)?

More Information

To ensure that your qPCR reactions are reproducible and reliable, follow our step-by-step design and optimization protocols. We also provide insights into analysis of qPCR data and troubleshooting reactions.

* Patent pending
** U.S. patents 6,627,424; 7,541,170; and 7,560,260.
*** US patent 9,493,824


Specifications

Product format Single-tube, ready-to use supermix
Concentration  4x concentrated
Detection chemistry Probes
Multiplex capability Up to 5 targets
Assembled reaction stability Up to 24 hours
Inhibitor tolerance Tested with ethanol and hematin. Also compatible with up to 10 µl of crude lysates
5’ to 3’ exonuclease activity of the enzyme(s) Yes
Hot-start, activation time Yes, 10 min
Sample input 1 pg to 1 µg
Sensitivity 10 copies in a 5-plex reaction
Reference dye Tented universal dye preblended in the supermix
Instrument compatibility Both ROX-dependent (high- and low-) and -independent instruments
Guaranteed shelf life at –20° 12 months
Shelf life at 4°C (after thawing) 1 week

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Reliance One-Step Multiplex RT-qPCR Supermix, 200 x 20 µl rxns, 1 ml

12010176
1 ml (1 x 1 ml vial), 4x one-step RT-qPCR supermix, contains Reliance Reverse Transcriptase, blend of high-performance DNA polymerases including Sso7d fusion polymerase, universal reference dye, stabilizer, and reaction buffer, for 200 x 20 μl rxns

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Reliance One-Step Multiplex Supermix, 1,000 x 20 µl rxns, 5 ml

12010220
5 ml (5 x 1 ml vials), 4x one-step RT-qPCR supermix, contains Reliance reverse transcriptase, blend of high-performance DNA polymerases including Sso7d fusion polymerase, universal reference dye, stabilizer, and reaction buffer, for 1,000 x 20 μl rxns

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$1,995.00 
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Reliance One-Step Multiplex Supermix, 2,000 x 20 µl rxns, 10 ml

12010221
10 ml (2 x 5 ml vials), 4x one-step RT-qPCR supermix, contains Reliance reverse transcriptase, blend of high-performance DNA polymerases including Sso7d fusion polymerase, universal reference dye, stabilizer, and reaction buffer, for 2,000 x 20 μl rxns

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SingleShot Cell Lysis Kit, 100 x 50 µl rxns

1725080
Pkg of 1, kit includes 5 ml (1 x 5 ml) of cell lysis buffer, 100 µl of proteinase K solution, and 100 µl of DNase solution, for 100 x 50 µl reactions

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$540.00 
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SingleShot Cell Lysis Kit, 500 x 50 µl rxns

1725081
Pkg of 1, kit includes 25 ml (5 x 5 ml) of cell lysis buffer, 500 µl of proteinase K solution, and 500 µl of DNase solution, for 500 x 50 µl reactions

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$2,393.00 
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CFX Automation System II for Real-Time PCR Systems

1845075
Plate handler and barcode scanner, mounting plate, automation software; does not include CFX Real-Time PCR Detection Systems

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Documents

Number Description Options
10000108800 Reliance One-Step Multiplex Supermix Product Insert Click to download
7197 Protocol for One-Step Multiplex RT-qPCR Analysis Using PrimePCR Probe Assays and Reliance One-Step Multiplex Supermix Click to download
7175 Reliance One-Step Multiplex Supermix Product Information Sheet, Ver B Click to download
7178 Amplification: Real-Time PCR Reagents and Kits Brochure Click to download
7196 Sensitive Codetection of Viral-Pathogen RNA and DNA Using One-Step Multiplex RT-qPCR Click to download
7275 Do More with Less: Five Steps to Great Results Using One-Step Multiplex RT-qPCR Click to download
10000112808 User Manual, Singleshot Cell Lysis Kit Click to download
6090 Amplification Reagents and Plastics Brochure Click to download [ Add to Cart (Free) ]
6252 Reagent Comparison Guide for Real-Time PCR Brochure, Rev C Click to download [ Add to Cart (Free) ]
6629 High-Throughput Real-Time PCR Solutions Brochure Click to download
6763 Gene Expression Real-Time PCR Workflow Flier Click to download

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