QuantiTect SYBR® Green RT-PCR Kit
For one-step qRT-PCR using SYBR Green I for gene expression analysis
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.
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.
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.
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.
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