商品名称：QuantiTect Probe RT-PCR Kit (1000)
QuantiTect RT-PCR Kits enable sensitive quantification of RNA targets by real-time one-step PCR using sequence-specific probes or SYBR Green I detection. The kits also allow reliable quantification of up to 5 RNA targets in a single tube by multiplex, real-time one-step RT-PCR. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive qRT-PCR on any real-time cycler without the need for optimization. The dNTP mix includes dUTP, allowing optional treatment with UNG. For convenience, the master mixes in QuantiTect one-step RT-PCR kits can be stored at 2–8°C.
Two kit formats are available for multiplex RT-PCR using sequence-specific probes: the QuantiTect Multiplex RT-PCR Kit for cyclers that require ROX dye for fluorescence normalization, and the QuantiTect Multiplex RT-PCR NoROX Kit for all other cyclers. The QuantiTect SYBR Green RT-PCR Kit is supplied with an optimized RT mix for efficient and sensitive reverse transcription over a wide range of RNA template amounts.
HotStarTaq DNA Polymerase included in QuantiTect one-step RT-PCR kits increases the specificity of the PCR reaction by providing the most stringent hot start compared with other polymerases.
The QuantiTect SYBR Green RT-PCR Kit contains a unique blend of reverse transcriptases, which delivers 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").
The QuantiTect Probe RT-PCR Kit has a unique RT-PCR buffer that promotes highly specific annealing of primers and probes to the PCR template. HotStarTaq DNA Polymerase and the unique composition of the RT-PCR buffer enable the QuantiTect Probe RT-PCR Kit to provide sensitive quantification of low-copy RNA targets, as well as accurate quantification over a wide linear range (see figure " High sensitivity and efficiency, and wide dynamic range").
Performing reverse transcription and PCR sequentially in the same tube does not impair sensitivity, as demonstrated by CT values that are comparable to those achieved in real-time two-step RT-PCR (see figures " One-step RT-PCR with comparable performance to two-step RT-PCR – A" and " One-step RT-PCR with comparable performance to two-step RT-PCR – B", and table “One-step RT-PCR with comparable performance to two-step RT-PCR”).
One-step RT-PCR with comparable performance to two-step RT-PCR.
| Amount of cDNA/RNA | Mean Ct value (two-step RT-PCR) | Mean Ct value (one-step RT-PCR) |
|---|---|---|
| 100 ng | 24.66 | 24.66 |
| 10 ng | 28.15 | 27.84 |
| 1 ng | 31.42 | 31.49 |
| 0.1 ng | 34.80 | 34.56 |
| 0.01 ng | 37.78 | 37.66 |
Sensitive detection of as few as 10 copies of target sequence (see figure " Detection of down to 10 copies of target RNA in duplex PCR") can be achieved in multiplex qRT-PCR with QuantiTect Multiplex RT-PCR Kits. Precise relative quantification of the expression of a gene is achieved by quantifying the expression of both the target gene and an endogenous control gene in the same well or tube. With QuantiTect Multiplex RT-PCR Kits, the CT values for the targets in a multiplex reaction are equivalent to those obtained in control experiments where the targets are amplified in separate reactions (see figure "Comparable amplification in triplex PCR and singleplex PCRs.
Triplex, real-time one-step RT-PCR was performed on the Applied Biosystems 7500 using the QuantiTect Multiplex RT-PCR Kit and TaqMan probes. The template was 20 ng total RNA from the Burkitt's lymphoma cell line Ramos. Reactions were performed in triplicate. 28S rRNA was detected using a HEX labeled probe. POLD3 (accessory subunit of DNA polymerase delta 3) was detected using a FAM labeled probe. CDK2 (cell cycle-dependent kinase 2) was detected using a Cy5 labeled probe. For comparison, the targets were also quantified by singleplex, real-time one-step RT-PCR (black curves). Curves for triplex PCR and singleplex PCRs overlap, demonstrating comparable amplification (i.e., equivalent CT values).
High sensitivity and efficiency, and a wide dynamic range.
One-step RT-PCR with comparable performance to two-step RT-PCR - A.
One-step RT-PCR with comparable performance to two-step RT-PCR - B.
Detection of down to 10 copies of target RNA in duplex PCR.
QuantiTect 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 or sequence-specific probes. The fluorescent dye SYBR Green I in the QuantiTect SYBR Green RT-PCR master mix enables the analysis of many different targets without having to synthesize target-specific labeled probes (see table “Components of 2x QuantiTect SYBR Green RT-PCR Kit”).
Components of 2x QuantiTect SYBR Green RT-PCR Kit
| Component | Features | 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 |
QuantiTect Probe RT-PCR Kits contain an optimized, ready-to-use master mix for highly specific and sensitive real-time quantification of RNA targets using sequence-specific probes (see table “Components of 2x QuantiTect Probe RT-PCR Kit”). The kits are designed for use with all types of sequence-specific probes, including hydrolysis probes (e.g., TaqMan® and other dual-labeled probes), FRET probes and Molecular Beacons.
Components of 2x QuantiTect SYBR Green RT-PCR Kit
| Component | Features | Benefits |
|---|---|---|
| HotStarTaq DNA Polymerase | 15 min activation at 95ºC | Set-up of qPCR reactions at room temperature |
| QuantiTect Probe 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 |
Precise relative quantification of the expression of a gene is achieved by quantifying the expression of both the target gene and an endogenous control gene in the same well or tube. The optimized master mix in the QuantiTect Multiplex RT-PCR Kit ensures that PCR products in a multiplex reaction are amplified with the same efficiency and sensitivity as PCR products in a corresponding single-amplification reactions (see table “Components of 2x QuantiTect Multiplex RT-PCR Kit”). As few as 10 copies of a target gene can be detected with the kit.
Components of 2x QuantiTect Multiplex RT-PCR Kit
| Component | Features | Benefits |
|---|---|---|
| HotStarTaq DNA Polymerase | 15 min activation at 95ºC | Set-up of qPCR reactions at room temperature |
| QuantiTect Multiplex RT-PCR Buffer | Balanced combination of NH4+ and K+ ions | Specific primer annealing ensures reliable PCR results |
| Synthetic Factor MP | Reliable multiplexing analysis of up to 4 genes in the same tube | |
| 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 |
| ROX dye* |
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