QuantiTect SYBR® Green RT-PCR Kit

For one-step qRT-PCR using SYBR Green I for gene expression analysis

  • High specificity in one-step RT-PCR
  • Reliable quantification of low-abundance transcripts
  • Accurate quantification over several logs of template
  • No need to optimize reaction and cycling conditions
The QuantiTect SYBR Green RT-PCR Kit provides highly specific quantification of RNA targets by real-time one-step RT-PCR using SYBR Green I detection. The combination of a hot start and a unique qRT-PCR buffer system ensures highly specific and sensitive real-time quantification of RNA targets. The dNTP mix includes dUTP, allowing optional treatment with UNG. The QuantiTect SYBR Green RT-PCR Kit is also supplied with an optimized RT mix for efficient and sensitive reverse transcription over a wide range of RNA template amounts. For convenience, the master mix can be stored at 2–8°C.
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QuantiTect SYBR Green RT-PCR Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect SYBR Green RT-PCR Master Mix, 100 µl QuantiTect RT Mix, 2 x 2 ml RNase-Free Water
204243
$1,752.00
QuantiTect SYBR Green RT-PCR Kit (1000)
For 1000 x 50 µl reactions: 25 ml 2x QuantiTect SYBR Green RT-PCR Master Mix, 0.5 ml QuantiTect RT Mix, 20 ml RNase-Free Water
204245
$6,859.00
The QuantiTect SYBR® Green RT-PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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One-step RT-PCR.|Highly specific amplification.|Comparable results in one-step and two-step RT-PCR.|Specific primer annealing.|
The QuantiTect SYBR® Green RT-PCR Kit overcomes the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers and template to the ready-to-use RT-PCR master mix, and start the reaction on any real-time cycler.|In comparison with other DNA polymerases, only HotStarTaq DNA Polymerase in combination with a unique buffer specifically amplified a 497 bp fragment (from 50 copies of an HIV-pol-gene construct in a background of 1 µg human genomic DNA). M: Markers.|Total RNA (10 ng to 100 pg) or the equivalent amounts of cDNA from HeLa cells were analyzed using the QuantiTect Primer Assay for human MAPK14 and the indicated kits. Similar CT values were achieved in one-step and two-step RT-PCR.|A balanced combination of KCl and (NH4)2SO4 promotes specific annealing of primers to the PCR template. K+ binds to phosphate groups on double-stranded DNA, stabilizing primer annealing. NH4+ destabilizes weak hydrogen bonds between mismatched bases.|
Performance
The QuantiTect SYBR Green RT-PCR Kit contains a unique blend of reverse transcriptases, which deliver efficient and sensitive cDNA synthesis over a wide range of amounts of RNA template and enables specific quantification over a wide linear range (see figure "Comparable results in one-step and two-step RT-PCR"). HotStarTaq DNA Polymerase further increases the specificity of the PCR reaction by providing the most stringent hot start compared with other polymerases (see figure "Highly specific amplification").
Principle

QuantiTect SYBR Green RT-PCR Kits contain an optimized, ready-to-use master mix for highly specific and sensitive real-time quantification of RNA targets using SYBR Green I. The fluorescent dye SYBR Green I in the master mix enables the analysis of many different targets without having to synthesize target-specific labeled probes. A balanced combination of K+ and NH4+ ions in the PCR buffer promotes specific primer annealing, enabling high RT-PCR specificity and sensitivity (see figure "Specific primer annealing"). In addition, an optimized mix of reverse transcriptases enables cDNA synthesis from a wide range of RNA template amounts, while  HotStarTaq DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.

QuantiTect SYBR Green RT-PCR master mix also contains dUTP, enabling pretreatment with uracil-N-glycosylase (UNG) prior to starting PCR, which ensures that any contaminating PCR products do not affect subsequent PCR reactions.

Components of 2x QuantiTect SYBR Green RT-PCR Kit
ComponentFeatures Benefits
HotStarTaq DNA Polymerase 15 min activation at 95ºC Set-up of qPCR reactions at room temperature
QuantiTect SYBR Green RT-PCR Buffer Balanced combination of NH4+ and K+ ions Specific primer annealing ensures reliable PCR results
dNTP mix Includes dUTP, which partially replaces dTTP and enables optional UNG treatment of reactions Eliminates contamination from carryover of PCR products by optional UNG treatment
SYBR Green I dye Yields a strong fluorescent signal upon binding double-stranded DNA Highly sensitive quantification
ROX dye For normalization of fluorescent signals on Applied Biosystems and, optionally,  Agilent instruments Precise quantification on cyclers that require ROX dye. Does not interfere with reactions on other real-time cyclers
Omniscript and Sensiscript Reverse Transcriptases Special blend of enzymes with high affinity for RNA RNA can be transcribed, even through complex secondary structures
Procedure

The QuantiTect SYBR Green RT-PCR Kit overcomes the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers and template to the ready-to-use RT-PCR master mix, and start the reaction (see flowchart "One-step RT-PCR"). Follow the protocol in the handbook to get fast and reliable results on any real-time cycler. If required, reactions can be pretreated with uracil-N-glycosylase (UNG) to eliminate carryover of PCR products from previous reactions. 

Highly specific results in gene expression analysis are guaranteed when QuantiTect SYBR Green RT-PCR Kits are used in combination with QuantiTect Primer Assays. These are genomewide, bioinformatically validated primer sets for detecting transcripts from human, mouse, rat, and many other species. QuantiTect Primer Assays can be easily ordered online at GeneGlobe.

Applications

The QuantiTect SYBR Green RT-PCR Kit is for use in gene expression analysis of RNA targets. QuantiTect SYBR Green RT-PCR Kits are compatible with all available real-time cyclers, including instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Agilent. For the Rotor-Gene Q and other Rotor-Gene cyclers, we recommend using the Rotor-Gene SYBR Green RT-PCR Kit, which has been specially developed for fast cycling on these instruments.

Feature
Specifications
Applications Real-time quantification of RNA targets
Reaction type Real-time and one-step RT-PCR
Real-time or endpoint Real-time
Sample/target type RNA
Single or multiplex Single
SYBR Green I or sequence-specific probes SYBR Green I
Thermal cycler All real-time cyclers (e.g. LC, RG, ABI)
With or without ROX With ROX

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Kit Handbooks
3
For genomewide, ready-to-use real-time RT-PCR assays using SYBR Green detection
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For quantitative, real-time one-step RT-PCR using SYBR Green I
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SYBR Green I を用いたリアルタイム定量1ステップRT-PCR 用
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