Advanced Molecular Diagnostics Ltd

SATO RT-qPCR Probe Master Mix

Catalogue Number KD4940881.1

Advanced Molecular Diagnostics Ltd is a diagnostics company

specialising in the manufacture and supply of molecular biology
instruments, reagents and consumables.

info@am-diagnostics.co.uk

+44 (0)115 969 9934

Address:
BioCity, Nottingham,
Pennyfoot Street,
Nottingham
NG1 1GF

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Contents
Kit Contents................................................................................................................................3
Overview ....................................................................................................................................3
Features .....................................................................................................................................4
Storage Specifications ................................................................................................................4
Basic Principles ...........................................................................................................................4
Protocols ....................................................................................................................................5
Additional Information ..............................................................................................................6
Additional equipment Required (not provided): .......................................................................6
Sample storage ..........................................................................................................................7
Sample transport: ......................................................................................................................7
Warnings and precautions: ........................................................................................................7
Product Use Limitations: ............................................................................................................7
Contact: ......................................................................................................................................7

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Kit Contents

1 x 1 ml SATO RT-qPCR Master Mix

1 x 0.1 ml RT Enzyme
1 x 1 ml DEPC Water

Overview
Sato RT-qPCR Probe Master Mix is a ready-to-use solution, optimized for real-time
quantitation of target RNA sequences using hydrolysis probes. One-step RT-qPCR provides a
convenient and powerful method for RNA detection and quantitation.

The Sato RT-qPCR Master Mix Kit for probe chemistry offers a quick and simple way to
detect mRNA. In a one-step approach, both the reverse transcription (RT) and qPCR steps
are performed in a single tube. Adopting a one-step approach is ideal when no cDNA
storage is necessary for subsequent experiments and minimises the risk of contamination
and improves reproducibility by reducing sample handling.

Probe-based quantitative RT-qPCR has a wide working temperature range (50-60 °C) and
improved success with GC-rich and other difficult templates, using real-time fluorescence
released upon 5´→3´ exonuclease cleavage of a quenched, target-specific probe to measure
the target gene amplification at each cycle of a PCR. When the fluorescence signal is
significantly detectable over the background fluorescence, the quantification cycle or
Cq value can be determined. Cq values can be used to evaluate relative target abundance
between two or more samples or to calculate absolute target quantities with reference to
an appropriate standard curve, derived from a series of known dilutions.

Using the fluorescence released during each of the reaction cycles, the expression of the
gene of interest can be measured.

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The master mix is formulated with a unique passive reference dye that is compatible across
a variety of instrument platforms, including those that require a high or low ROX reference
signal.

Features
• Efficient– Using multiple probes in the master mix allows you detect to multiple amplicons
(fluorophore signals) in the same tube.
• Accurate– we ensure all of our probes have high specificity to the target area.
• High yield–our buffer ensures that start Taq DNA polymerase performs at its best to
maximise DNA yield.
• Robust– High fidelity amplification with little optimisation required.

Storage Specifications
The kits should be transported and stored at temperatures between -15 °C and -30 °C. The
kit will remain stable at least until the expiry date printed on the package, if the storage
temperature is kept. Repeated freezing and thawing of the kit components may result in
lower detection quality.

Basic Principles
The RT-qPCR Kit is a real-time one-step reverse transcription PCR reaction. It is designed to
increase speed, higher inhibitor tolerance, and optimize PCR efficiency. From gene
expression analysis, population genotyping studies, to the multiplex detection of several
targets real-time PCR with excellent fast, sensitivity and specificity.

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Protocols
1. Thaw all reagents and store on ice. Mix and centrifuge all reagents well before
preparing the master mix. Check for precipitation

2. Combine all reagents together in a nuclease free eppendorf, using the table 1 as a
guide. Mix thoroughly and briefly centrifuge.

Table 1: Reagents required per sample

Component Amount
RT Enzyme Mix (20X) 1 μl
SATO RT-qPCR Master Mix 10 μl
2X
Forward primers 20X (200 1 μl
nM) (Up to 1 μM each)
Reverse primers 20X (200 1 μl
nM) (Up to 1 μM each)
Nuclease free water To 20 μl
RNA template Volume varies
(Up to 250ng)

3. Aliquot the master mix into separate 0.2 mL PCR tubes before adding the DNA
template.
4. Gently mix together and centrifuge briefly.
5. Use Table 2 as a guide to set up the thermal profile, considering the melting
temperature of the primers. Set ROX as the reference dye.
6. Add and incubate reactions into the qPCR thermocycler
7. Once the reaction is complete, Ct value data can be analysed for gene expression, using
the appropriate programme.

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 Table 2: Temperature and Times for the PCR protocol
Stage Temperature Time
Stage 1 50-55°C 15 mins
Reverse Transcription
Stage 2 95°C 5 mins
Enz. Activation
Stage 3.1 95°C 10 sec
Denaturation
Stage 3.2 60°C *30 sec
Annealing/Extension
Repeat stage 3 for 40 cycles
*Increase to 1min, if GC content is ≤50%

Additional Information

Tm calculation – For primers below 25 nucleotides, please use this equation to calculate the Tm:

Tm = 4(G+C) + 2(A+T)

If the primer has more than 25 nucleotides this equation is insufficient, and computational
methods are required.

Template – Use up to 250ng of template RNA to minimise errors in the product.

Additional equipment Required (not provided):

Total RNA: The SATO RT-qPCR kit can be used with RNA of up to 250ng.

Specific Gene primers: With this RT-qPCR kit, no other reagents are required other than
the specific forward and reverse primers specific to the gene of interest.

Disposables: AMD manufacture high quality PCR plastic wear suitable for use with this
kit. It is also recommended that correct PPE including gloves is used throughout.

Other Laboratory Equipment: qPCR Thermocycler, Vortex, centrifuge, pipettes and tips.

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Sample storage:
As a general rule, multiple freeze-thaws should be avoided. The most practical way to address this
concern is by aliquoting samples after collection.

Sample transport:
Sample material should be transported in a shatterproof transport container as a matter of principle.
Thus, a potential danger of infection due to a leakage of sample can be avoided. The samples should
be transported following the local and national instructions for the transport of pathogen material.
The samples should be shipped within 6 hours. It is not recommended to store the samples where
they have been collected. It is possible to ship the samples by mail, following the legal instructions
for the transport of pathogen material. We recommend the sample transport with a courier.

Warnings and precautions:

When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective
goggles. For more information, please consult the appropriate safety data sheets (SDSs). Discard
sample and assay waste according to your local safety regulations.

Product Use Limitations:

All reagents may exclusively be used in in vitro diagnostics. The product is to be used by personnel
specially instructed and trained in the in vitro diagnostics procedures only. Strict compliance with
the user manual is required for optimal PCR results. Attention should be paid to expiration dates
printed on the box and labels of all components. Do not use expired components.

Contact:
Any queries, comments or complaints please refer to our website at:

www.am-diagnostics.co.uk

info@am-diagnostics.co.uk