Agilent Brilliant II QRT-PCR Master Mix Kit User Guide
Brilliant II QRT-PCR Master Mix Kit, 1-Step
Instruction Manual
Catalog #600809 (single kit)
#600818 (10-pack kit)
Revision C
Research Use Only. Not for Use in Diagnostic Procedures.
600809-12
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this product.
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Brilliant II QRT-PCR Master Mix Kit, 1-Step
CONTENTS
Materials Provided.............................................................................................................................. 1
Storage Conditions.............................................................................................................................. 1
Additional Materials Required .......................................................................................................... 1
Notices to Purchaser ........................................................................................................................... 1
Introduction......................................................................................................................................... 2
Features of Kit Components.................................................................................................. 2
Molecular Beacons Probes .................................................................................................... 3
TaqMan® Probes (Hydrolysis Probes)................................................................................... 3
Fluorescence Monitoring in Real-Time................................................................................. 5
Preprotocol Considerations................................................................................................................ 7
RNA Isolation........................................................................................................................ 7
Quantitative PCR Human Reference Total RNA .................................................................. 7
Probe Design ......................................................................................................................... 8
Optimal Concentrations for Experimental Probes and PCR Primers .................................... 8
Reference Dye ....................................................................................................................... 9
Magnesium Chloride Concentration...................................................................................... 9
Preparing a Single Mixture for Multiple Samples................................................................. 9
Mixing and Pipetting Enzymes ........................................................................................... 10
Temperature and Duration of cDNA Synthesis Reaction.................................................... 10
Preventing Sample Contamination ...................................................................................... 10
Recommended Control Reactions ....................................................................................... 10
Endpoint vs. Real-Time Measurements............................................................................... 11
Data Acquisition with a Spectrofluorometric Thermal Cycler............................................ 11
Multiplex RT-PCR .............................................................................................................. 11
Protocol .............................................................................................................................................. 13
Preparing the Reactions....................................................................................................... 13
RT-PCR Cycling Programs ................................................................................................. 15
Troubleshooting: TaqMan® Probes................................................................................................. 16
Troubleshooting: Molecular Beacons..............................................................................................17
References .......................................................................................................................................... 18
Endnotes............................................................................................................................................. 18
MSDS Information............................................................................................................................ 18
Quick-Reference Protocol ................................................................................................................ 19
Brilliant II QRT-PCR Master Mix Kit, 1-Step
ATERIALS PROVIDED
M
Catalog #600809 (single kit), #600818 (10-pack kit)
Materials provided Quantity
2× Brilliant II QRT-PCR Master Mix 2 × 2.5 ml
RT/RNase Block Enzyme Mixture 400 μl
Reference dye c, 1 mM 100 μl
a
Sufficient PCR reagents are provided for four hundred, 25-μl QRT-PCR reactions
b
Quantities listed are for a single kit. For 10-pack kits, each item is provided at 10 times the listed quantity.
c
The reference dye is light sensitive and should be kept away from light whenever possible.
a,b
STORAGE CONDITIONS
All Components: Upon receipt, store all components at –20°C. Store the 2× master mix at 4°C after
thawing. Once thawed, full activity is guaranteed for 6 months.
Note The reference dye is light sensitive and should be kept away from light whenever possible.
ADDITIONAL MATERIALS REQUIRED
Spectrofluorometric thermal cycler
Nuclease-free PCR-grade water
NOTICES TO PURCHASER
Notice to Purchaser: Limited License
Practice of the patented 5’ Nuclease Process requires a license from Applied Biosystems. The
purchase of this product includes an immunity from suit under patents specified in the product insert
to use only the amount purchased for the purchaser's own internal research when used with the
separate purchase of Licensed Probe. No other patent rights are conveyed expressly, by implication,
or by estoppel. Further information on purchasing licenses may be obtained from the Director of
Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
Revision C © Agilent Technologies, Inc. 2010.
Brilliant II QRT-PCR Master Mix Kit, 1-Step 1
INTRODUCTION
Quantitative PCR is a powerful tool for gene expression analysis. Many
fluorescent chemistries are used to detect and quantitate gene transcripts.
The use of fluorescent probe technologies reduces the risk of sample
contamination while maintaining convenience, speed, and high-throughput
screening capabilities. The Brilliant II QRT-PCR Master Mix Kit, 1-Step
can be used with both hairpin and linear fluorescent probe technologies to
perform absolute or relative quantitation of gene expression. The single-step
master mix format is ideal for most high-throughput QPCR applications
where it is not necessary to archive cDNA.
The Brilliant II QRT-PCR master mix kit includes the components
necessary to carry out cDNA synthesis and PCR amplification in one tube
and one buffer.* Brilliant kits support quantitative amplification and
detection with multiplex capability and show consistent high performance
with various fluorescent detection systems, including molecular beacons and
®
TaqMan
probes. The Brilliant II QRT-PCR master mix kit has been
successfully used to amplify and detect a variety of high- and lowabundance RNA targets from experimental samples including total RNA,
+
poly(A)
RNA, and synthetic RNA.
The Brilliant II QRT-PCR master mix has been optimized for maximum
performance on the Stratagene Mx3000P and Mx3005P real-time PCR
systems and the Stratagene Mx4000 multiplex quantitative PCR system, as
well as on the ABI 7900HT real-time PCR instrument.
Features of Kit Components
RT/RNase Block Enzyme Mixture
The reverse transcriptase (RT) provided in the kit is a Moloney-based RT
specifically formulated for Stratagene Brilliant II kits. This RT performs
optimally at a reaction temperature of 50°C when used in 1-step QRT-PCR
with the Brilliant II master mix. It is stringently quality-controlled to verify
the absence of nuclease contaminants that adversely affect cDNA synthesis
and to ensure sensitive and reproducible performance in QRT-PCR
experiments with a broad range of RNA template amounts and a variety of
RNA targets that vary in size, abundance, and GC-content. The RNase
block, provided in the same tube, serves as a safeguard against
contaminating RNases.
* Primers and template are not included.
2 Brilliant II QRT-PCR Master Mix Kit, 1-Step
Brilliant II QRT-PCR 2× Master Mix
The 2× master mix contains an optimized RT-PCR buffer, MgCl2,
nucleotides (GAUC), stabilizers, and SureStart Taq DNA polymerase.
SureStart Taq DNA polymerase is a modified version of Taq2000 DNA
polymerase with hot start capability. SureStart Taq DNA polymerase
improves PCR performance by decreasing background and increasing
amplification of desired products. Using SureStart Taq, hot start is easily
incorporated into PCR protocols already optimized with Taq DNA
polymerase, with little or no modification of cycling parameters or reaction
conditions.
Reference dye
A passive reference dye (an optional reaction component) is provided in a
third tube. The passive reference dye (with excitation and emission
wavelengths of 584 nm and 612 nm, respectively) is provided as an optional
reagent that may be added to compensate for non-PCR related variations in
fluorescence. Providing the reference dye in a separate tube makes the
Brilliant II QRT-PCR master mix kit adaptable for many real-time QPCR
platforms (see Reference Dye in Preprotocol Considerations for more
information).
Molecular Beacons Probes
Molecular beacons are hairpin-shaped fluorescent hybridization probes that
can be used to monitor the accumulation of specific product during or after
1–5
PCR.
opposite ends of an oligonucleotide (see Figure 1). The ends of the
oligonucleotide are designed to be complementary to each other. When the
unhybridized probe is in solution, it adopts a hairpin structure that brings the
fluorophore and quencher sufficiently close to each other to allow efficient
quenching of the fluorophore. If, however, the molecular beacon is bound to
its complementary target, the fluorophore and quencher are far enough apart
that the fluorophore cannot be quenched and the molecular beacon
fluoresces. As PCR proceeds, product accumulates and the molecular
beacon fluoresces at a wavelength characteristic of the particular
fluorophore used. The amount of fluorescence at any given cycle depends
on the amount of specific product present at that time.
Molecular beacons have a fluorophore and a quencher molecule at
TaqMan® Probes (Hydrolysis Probes)
TaqMan probes are linear.
probe, and the quencher is either internal or is at the 3´ end (see Figure 2).
As long as the probe is intact, regardless of whether it is hybridized with the
target or free in solution, no fluorescence is observed from the fluorophore.
During the combined annealing-extension step of PCR, the primers and the
TaqMan probe hybridize with the target. The DNA polymerase displaces the
TaqMan probe by 3 or 4 nucleotides, and the 5´-nuclease activity of the
DNA polymerase separates the fluorophore from the quencher. Because of
this mechanism of action, these probes are also referred to as hydrolysis
probes. Fluorescence can be detected during each PCR cycle, and
fluorescence accumulates during the course of PCR.
Brilliant II QRT-PCR Master Mix Kit, 1-Step 3
6, 7
The fluorophore is usually at the 5´ end of the
A
e
,
Fluorophore
Quencher
Molecular
beacon
FIGURE 1 The molecular beacon binds to a complementary target and fluoresces.
Ta rg et
Hybrid
PCR Primer
PCR Primer
Fluorophor
Polymerization
Ta q Ma n
probe
mplification assay
Quencher
PCR Primer
- - - - - - - -
- - - - - -
Probe displacement
and cleavage
Fluorescence
- - - - - - - - - - -
- - - - - - - - - -
Result
Fluorescence
PCR products
FIGURE 2 TaqMan probe fluoresces when the 5´-nuclease activity of the DNA polymerase separates the fluorophore from
quencher.
4 Brilliant II QRT-PCR Master Mix Kit, 1-Step
Cleavage products
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