UltraPlex® 1-Step ToughMix® ROX™ (4X)
Cat No.

95167-100Size:

100 x 20 µL reactions (500 µL)

Store at -25ºC to -15°C
protected from light

Description
UltraPlex 1-Step ToughMix ROX is a ready-to-use, 4X-concentrated master mix for reverse transcription quantitative PCR (RT-qPCR) of RNA templates
using hybridization probe detection chemistries such as TaqMan® 5’-hydrolysis probes on real-time PCR systems that utilize ROX passive reference with
490 nM excitation. First-strand cDNA synthesis and PCR amplification are carried out in the same tube without opening between procedures. It is ideal forhighly sensitive quantification of RNA viruses or low abundance RNA targets in uni- or multiplexed RT-qPCR applications as well as high throughput geneexpression studies. The system has been optimized to deliver maximum RT-qPCR efficiency, sensitivity, and specificity in reduced reaction volumes andfast cycle times. UltraPlex 1-Step ToughMix ROX contains all required components for RT-qPCR except RNA template and probe. It is compatible with all
dual-labeled probe chemistries.
qScript® XLT is an engineered M-MLV reverse transcriptase (RT) with reduced RNase H activity for improved activity and stability at higher temperatures.
The use of higher temperatures (50 to 54°C) for the first-strand step of one-step RT-qPCR provides higher specificity for primer annealing and disruption ofRNA secondary structure that can interfere with cDNA synthesis. These beneficial properties of qScript XLT RT are further enhanced by a hot-startmechanism for the reverse transcription step. Minimizing off-target extension by the RT during reaction assembly provides highly reproducible low copy
quantification as well as extended room temperature stability of fully assembled reactions for high throughput operations.
UltraPlex 1-Step ToughMix ROX is highly resistant to PCR inhibitors. A key component of the ToughMix is an ultra pure, highly processive thermostable
DNA polymerase that is combined with high avidity monoclonal antibodies. This provides an extremely stringent automatic hot-start that minimizes thepotential for primer-dimer and other non-specific PCR artifacts.
Instrument Compatibility
Different real-time PCR systems employ different strategies for the normalization of fluorescent signals and correction of well-to-well optical variations. It iscritical to match the appropriate qPCR reagent to your specific instrument. UltraPlex 1-Step ToughMix ROX provides seamless integration on the Applied
Biosystems 7000, 7300, 7700, 7900HT StepOne™, or StepOnePlus™. Please visit our web site at www.quantabio.com to find an optimized kit for yourinstrument platform(s).
Components
Reagent
UltraPlex 1-Step ToughMix ROX (4X)

Description
4X reaction buffer containing dATP, dCTP, dGTP, dTTP, magnesium, qScript XLT reversetranscriptase, RNase inhibitor protein, hot-start DNA polymerase, stabilizers, and ROX
Reference Dye

Storage and Stability

Store components in a constant temperature freezer at -25°C to -15°C protected from light upon receipt.
For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form.
Guidelines for One-Step RT-qPCR
The design of highly specific primers and probes is a critical parameter for successful one-step RT-qPCR. The use of computer aided primer

design programs is encouraged in order to minimize the potential for internal secondary structure and complementation at 3’-ends withineach primer, the primer pair, and primer/probe combinations. Regions of strong RNA secondary structure should be avoided as this caninterfere with primer hybridization and/or impede procession of the reverse transcriptase. For best results, amplicon size should be between
70 and 150 bp. Optimal results may require titration of primer concentration between 400 and 900 nM. A final concentration of 450 nM
each primer and 100 to 150 nM probe is effective for most applications. The efficacy and efficiency of any primer/probe set should bevalidated under fast cycling and/or rapid ramp rate protocols before use in qPCR studies.

95167 / IFU-110.1 REV 04

1

Guidelines for One-Step RT-qPCR continued:
If frozen, thaw UltraPlex 1-Step ToughMix ROX on ice. Thoroughly mix by vortexing, and then centrifuge to collect contents to the bottom of thetube. Retain on ice before use.
First-strand synthesis can be carried out between 42°C and 60°C. Optimal results are generally obtained with a 10-minute incubation at 50°C.
Longer incubation times for first-strand synthesis (up to 20 min) may be used. Incubation at temperatures over 54°C may result in delayed Cqs forassays that are optimal at 48 - 50°C.
We recommend a minimum of 30s incubation at 95°C to inactivate the RT and activate the hot-start polymerase prior to PCR cycling. Longeractivation times (2 – 10 minutes) will not adversely affect product performance and may reduce early cycle background noise experienced withsome hydrolysis probe chemistries.
The kit is compatible with both fast or standard qPCR cycling protocols. Annealing and or extension temperatures may need to be optimized for agiven primer/probe design or fluorogenic probe chemistry. Use the suggested protocol as a starting point. Multiplexed RT-qPCR may benefit froma slightly longer extension time (60 to 90s). Use of a slower ramp rate (~2.5°C/s) between the denaturation step and annealing/extension step may
improve performance for some assays.
To maximize specificity, reactions should be assembled and retained on ice before transfer to the qPCR instrument.
Preparation of a reaction cocktail is recommended to reduce pipetting errors and maximize assay precision. Assemble the reaction cocktail with all
required components except RNA template and mix thoroughly by vortexing. Then, dispense equal aliquots into each reaction tube. Add RNA
to each reaction as the final step. Addition of sample as 2 to 5-µL volumes will improve assay precision.
Suggested input quantities of template are: 1 pg to 100 ng total RNA; 10 fg to 10 ng poly A(+) RNA; 10 to 1x108 copies viral RNA.
After sealing each reaction, mix contents by vortexing, and then centrifuge briefly to collect components at the bottom of the reaction tube.
Reaction AssemblyComponent
Volume for 20-μL rxn.
UltraPlex 1-Step ToughMix ROX (4X)
5 µL
Forward primer(s)variable
Reverse primer(s)variable
Probe(s)variable
Nuclease-free water
variable
RNA template
2 to 10 µL
Final Volume (μL)
20 µL
Note: For smaller, or larger, reaction volumes scale all components proportionally.

Final Concentration
1X
300 – 900 nM
300 – 900 nM
50-200 nM
variable

RT-qPCR Cycling Protocol
Incubate complete reaction mix in a real-time PCR detection system as follows:
cDNA Synthesis
50°C, 10 min
Initial denaturation
95°C, 3 min
PCR cycling (30 - 45 cycles)
95°C, 3s to 10s
60°C, 30s to 90s (data collection step)‡‡Note: The use of longer extension times (90s at 60°C), or a 3-step cycling protocol with an extension step of 60s at 72°C can help mitigate suppressionof a low copy amplicon when co-amplified with a high copy target sequence.

Quality Control
Kit components are free of contaminating DNase and RNase. UltraPlex 1-Step ToughMix ROX is functionally tested in duplexed RT-qPCR. Kinetic
analysis must demonstrate linear resolution over six orders of dynamic range (r2 ≥ 0.990) and a PCR efficiency ≥ 90% for the primary GOI with constantdetection for the limiting exogenous positive control assay.

Limited Label Licenses

Use of this product signifies the agreement of any purchaser or user of the product to the following terms:
1. The product may be used solely in accordance with the protocols provided with the product and this manual and for use with components contained in the kit
only. Quantabio, LLC. grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included
within this kit except as described in the protocols provided with the product, this manual, and additional protocols available at www.quantabio.com. Some of these
additional protocols have been provided by Quantabio product users. These protocols have not been thoroughly tested or optimized by Quantabio, LLC. Quantabio,LLC. neither guarantees them nor warrants that they do not infringe the rights of third-parties.
2. Other than expressly stated licenses, Quantabio, LLC. makes no warranty that this kit and/or its use(s) do not infringe the rights of third-parties.
3. This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.
4. Quantabio, LLC. specifically disclaims any other licenses, expressed or implied other than those expressly stated.
5. The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. Quantabio, LLC.
may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action
to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components.
©2021 Quantabio, LLC. 100 Cummings Center Suite 407J Beverly, MA 01915; Telephone number: 1-888-959-5165.
Quantabio products are manufactured in Beverly, Massachusetts, Frederick, Maryland and Hilden, Germany.
Intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.
UltraPlex, ToughMix and qScript are registered trademarks of Quantabio, LLC. TaqMan is a registered trademark of Roche Molecular Systems, Inc.. ROX is a trademark of Life Technologies
Corporation.

95167 / IFU-110.1 REV 042