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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Product		Cat. no.	List price:
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	Inquire
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	Inquire

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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 C_T 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
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

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

Brochures & Guides

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	Flexible RNAi Technologies You Can Rely On - (EN)	Show details
	(EN) - Analyzing Gene Expression and Regulation	Show details
	Critical Factors for Successful Real-Time PCR - (EN)	Show details

FAQs

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	Do you recommend 1-step or 2-step real-time RT-PCR for gene expression analysis? FAQ ID -1056	View
	Why does my PCR product show up later when comparing the QuantiTect SYBR Green PCR Kits with Roche kits using the same annealing temperature? FAQ ID -1083	View
	Why do melting temperatures differ between PCR fragments amplified with QIAGEN's QuantiTect SYBR Green PCR Kits and Roche Kits? FAQ ID -1084	View
	What should I use as a standard for absolute quantification in real-time PCR? FAQ ID -1085	View
	How much template can I use in the reaction and what is the maximum volume of template that can be used in the QuantiTect Kits? FAQ ID -1086	View
	What is a QuantiTect Primer Assay? FAQ ID -1141	View
	Do QuantiTect Primer Assays contain SYBR Green dye? FAQ ID -1143	View
	Can I increase the annealing temperature recommended for QuantiTect Primer Assays used with the QuantiTect SYBR Green PCR Kits? FAQ ID -1146	View
	How much time will be saved when switching from standard cycling to fast cycling with QuantiFast Kits? FAQ ID -1438	View
	How do QuantiFast PCR Kits compare to QuantiTect PCR Kits for quantitative real-time PCR? FAQ ID -1441	View
	Why is the storage time for QuantiFast PCR Kits shorter than that for QuantiTect PCR Kits? FAQ ID -1446	View
	Why is the reaction volume for QuantiFast PCR Kits lower than that for QuantiTect PCR Kits? FAQ ID -1447	View
	Why is the RT step with the QuantiFast RT Kits much shorter compared to QuantiTect RT Kits? FAQ ID -1451	View
	Have QuantiTect Primer Assays been tested with QuantiFast SYBR Green PCR Kits on the Mastercycler ep realplex? FAQ ID -1714	View
	QuantiTect Primer Assays are bioinformatically validated, genomewide primer sets. What does “bioinformatically validated” mean? FAQ ID -1982	View
	Why is an ice-incubation step included during reaction set-up when following the QuantiTect RT-PCR but not the QuantiTect PCR protocol. FAQ ID -283	View
	Can Uracil-N-Glycosylase (UNG) be purchased separately, independent of QuantiTect PCR Kits? FAQ ID -2134	View
	Can Uracil-N-Glycosylase (UNG) be used with a one-step RT-PCR kit, such as the QuantiTect Probe RT-PCR Kit? FAQ ID -2135	View
	Do you have any information or guidelines regarding the choice of reference genes for real-time PCR? FAQ ID -2371	View
	How do I create a workspace that is free of DNA contamination, prior to carrying out a qPCR experiment? FAQ ID -2654	View
	How important is the RNA purification process, for obtaining reliable qRT-PCR results? FAQ ID -2655	View
	What is the threshold cycle or Ct value? FAQ ID -2682	View
	Does SYBR Green dye present in QuantiTect kits interfere with ethidium bromide staining? FAQ ID -312	View
	Do the QuantiTect SYBR Green Kits contain a stabilization reagent that inhibits the sensitivity of real-time PCR? FAQ ID -328	View
	My QuantiTect Master Mix did not freeze at degrees. Is it still ok to use? -20	View
	Why do replicates in real-time PCR have different plateau heights? FAQ ID -539	View
	What reaction volume should I use with QuantiTect Kits? FAQ ID -551	View
	How long should the amplicon be if I am using QuantiTect SYBR Green detection? FAQ ID -553	View
	Is it possible to perform a UNG treatment when using QuantiTect kits? FAQ ID -564	View
	Do you have information on the use of recombinant DNA and RNA as absolute standards for realtime RT-PCR? FAQ ID -729	View
	Why should DNA or cDNA targets be less than 250 bp long for real-time PCR? FAQ ID-751	View
	What annealing temperature should be used with the QuantiTect Primer Assays? FAQ ID -849	View
	What is the concentration of the primers in a reaction using the QuantiTect Primer Assays? FAQ ID -850	View

Scientific Posters

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	Quantitative RT-PCR analysis with genomewide, validated primer sets - (EN)	Show details

Kit Handbooks

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	(EN) - QuantiTect Primer Assay Handbook For genomewide, ready-to-use real-time RT-PCR assays using SYBR Green detection	Show details
	(EN) - QuantiTect SYBR Green RT-PCR Handbook For quantitative, real-time one-step RT-PCR using SYBR Green I	Show details

Safety Data Sheets

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	MSDS QuantiTect SYBR Green RT-PCR Kit (200)
	MSDS QuantiTect SYBR Green RT-PCR Kit (1000)
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