QuantiTect Reverse Transcription Kit

Sensitive real-time PCR을 two-step RT-PCR로 할 때 cDNA 합성을 신속히 할 수 있습니다

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QuantiTect Rev. Transcription Kit (50)

Cat. No. / ID: 205311

For 50 x 20 µl reactions: 100 µl 7x gDNA Wipeout Buffer, 50 µl Quantiscript Reverse Transcriptase, 200 µl 5x Quantiscript RT Buffer, 50 µl RT Primer Mix, 1.9 ml RNase-Free Water

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400

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QuantiTect Reverse Transcription Kit은/는 분자생물학 분야에 사용하기 위한 것입니다. 이 제품은 질병의 진단, 예방, 또는 치료용이 아닙니다.

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특징

빠르고 편리한 과정 — 단지 20분만에 genomic DNA를 제거하고 cDNA 합성
High sensitivity — obtain high cDNA yields even from low-abundance transcripts
RT from all regions — reliably detect any target in real-time RT-PCR, even those from 5' regions
효율적인 genomic DNA 제거 — RNA-specific primer 혹은 probe 디자인 필요하지 않음

제품 세부 정보

The unique QuantiTect Reverse Transcription Kit provides a fast and convenient procedure for cDNA synthesis with integrated genomic DNA removal. Genomic DNA contamination in RNA samples is effectively eliminated by gDNA Wipeout Buffer. All components that are required for fast and efficient reverse transcription are provided with the QuantiTect Reverse Transcription Kit, including Quantiscript Reverse Transcriptase, Quantiscript RT Buffer, and a unique RT Primer Mix. The synthesized cDNA is optimized for use in real-time PCR, allowing reliable quantification of targets from all regions of mRNA transcripts.

성능

Using the QuantiTect Reverse Transcription Kit, contaminating genomic DNA in RNA samples is effectively and rapidly removed with the unique gDNA Wipeout Buffer (see figure " Effective genomic DNA removal for accurate real-time RT-PCR"). Elimination of genomic DNA is crucial for accurate gene expression results, and design of RNA-specific primers or probes is not always possible. With gDNA Wipeout Buffer, time is saved and costs are reduced, since a separate DNase digestion is unnecessary, either during or after purification of RNA samples.

The high RNA affinity of Quantiscript Reverse Transcriptase, in combination with Quantiscript RT Buffer, enables high yields of cDNA from any RNA template (see table “Higher cDNA yields for less abundant transcripts”). Even difficult templates, such as those with high GC-content or complex secondary structure, are successfully reverse transcribed.

Higher cDNA yields for less abundant transcripts
	C_T values for IL12A (low expression)		C_T values for IL1RN (higher expression)
Input RNA (ng)	QIAGEN	Supplier A_II	QIAGEN	Supplier A_II
1000	30.9	32.0	23.1	24.9
100	34.2	35.4	26.3	26.6
10	37.8	46.8	29.7	30.3
1	N.D.	N.D.	32.4	34.5

The RT Primer Mix contains a specially optimized mix of oligo-dT and random primers that enable cDNA synthesis from all regions of RNA transcripts, even from 5' regions (see figure " Sensitive detection of a target at the 5' region of a 12.5 kb transcript"). In contrast to kits from other suppliers, the QuantiTect Reverse Transcription Kit provides high yields of cDNA template for real-time PCR analysis regardless of where the amplified target region is located on the transcript, and provides greater sensitivity in the detection of low-abundance genes (see figure " Higher sensitivity in real-time, two-step RT-PCR"). The QuantiTect Reverse Transcription Kit also enables greater reproducibility in real-time RT-PCR.

그림 참조

Effective genomic DNA removal for accurate real-time RT-PCR.

Sensitive detection of a target at the 5' region of a 12.5 kb transcript.

Higher sensitivity in real-time, two-step RT-PCR.

원리

QuantiTect Reverse Transcriptase is a novel blend of Omniscript and Sensiscript Reverse Transcriptases, which has a high affinity for RNA and is capable of cDNA synthesis from a wide range of RNA amounts (10 pg to 1 µg). In contrast to kits from other suppliers, the QuantiTect Reverse Transcription Kit provides high yields of cDNA template for real-time PCR analysis regardless of where the amplified target region is located on the transcript. Even difficult templates, such as those with high GC-content or complex secondary structure, are successfully reverse transcribed. QuantiTect RT Buffer has also been optimized to be compatible with real-time PCR buffer.

To obtain accurate results in real-time RT-PCR gene expression assays, it is important that only cDNA is amplified and detected. Interference by genomic DNA can be avoided by designing primers or probes that span an exon/exon boundary. However, in cases where this is not possible (e.g., the cDNA is from a single-exon gene), it is essential that the starting RNA sample is free of genomic DNA. Using the QuantiTect Reverse Transcription Kit, contaminating genomic DNA in RNA samples is effectively and rapidly removed with unique gDNA Wipeout Buffer. Time is saved and costs are reduced, since a separate DNase digestion not required, either during or after purification of RNA samples. Also, design of RNA-specific primers or probes is unnecessary.

Components of the QuantiTect Reverse Transcriptase Kit
Component	Benefits
gDNA Wipeout Buffer	Detection of RNA only in real-time RT-PCR
Quantiscript Reverse Transcriptase	Use of a wide range of RNA amounts (10 pg to 1 µg RNA) High sensitivity
Quantiscript RT Buffer	Read-through of difficult templates
RT Primer Mix	cDNA synthesis from all regions of transcripts, even from 5' regions

절차

Genomic DNA removal and cDNA synthesis take only 20 minutes with the QuantiTect Reverse Transcription Kit (see flowchart " Fast and convenient cDNA synthesis"). The procedure is fast and convenient, since both reactions are run using the same incubation temperature and are set up using master mixes.

The QuantiTect Reverse Transcription Kit includes everything you need for fast cDNA synthesis. Purified RNA is briefly incubated in gDNA Wipeout Buffer to effectively remove contaminating genomic DNA. In contrast to other methods, the RNA sample is then used directly in reverse transcription, using a master mix prepared from Quantiscript Reverse Transcriptase, Quantiscript RT Buffer, and RT Primer Mix. With Quantiscript Reverse Transcriptase, RNA can be transcribed at low temperatures, even through complex 2° structure, ensuring that the RNA will stay intact — the entire reaction takes place at 42°C and is then inactivated at 95°C. Additional steps for RNA denaturation, primer annealing, and RNase H digestion are not necessary.

그림 참조

Fast and convenient cDNA synthesis.

응용 분야

The QuantiTect Reverse Transcription Kit allows highly efficient and sensitive real-time RT-PCR for all types of starting material, including laser-microdissected samples and tissue biopsies.

지원되는 데이터 및 수치

Fast and convenient cDNA synthesis.

Genomic DNA removal and cDNA synthesis take only 20 minutes with the QuantiTect Reverse Transcription Kit. The procedure is fast and convenient since both reactions are run using the same incubation temperature and are set up using master mixes. In contrast, the procedure for the kit from Supplier I is much longer and requires more "hands-on time" due to additional pipetting steps and frequent changes in incubation temperature.

Specifications

Features	Specifications
Applications	Quantification of (even low-abundance) transcripts
Sample/target type	RNA template
Enzyme activity	Reverse transcription
Real-time or endpoint	Real time
Reaction type	Two-step, cDNA production, genomic DNA digestion
Single or multiplex	Single
Mastermix	No

리소스

For cDNA synthesis with integrated removal of genomic DNA contamination For use in real-time two-step RT-PCR

FAQ

Ensure PCR amplicons are as short as possible, ideally 60–150 bp. Always use the same algorithm or software to design the primers and probes. For optimal results, only combine assays that have been designed using the same parameters.

Check the functionality of each set of primers and probes in individual assays before combining the different sets in the multiplex assay. Choose compatible reporters and quenchers based on a specific instrument. See How do I select appropriate reporter and quencher combinations for multiplex PCR.

FAQ ID -9093

Yes, it is possible to use the QuantiTect Reverse Transcription Kit for bacteria. The RT Primer Mix provided in the kit is a unique, optimized blend of random primers and oligo-dT allowing high cDNA yields from all regions of RNA transcripts. It has successfully been tested for Reverse Transcription in bacteria as well. We strongly recommend to isolate bacterial RNA using the RNeasy Mini Kit prior to performing Reverse Transcription. This will ensure the high prep quality necessary for optimal RT results with the QuantiTect Reverse Transcription Kit.

FAQ ID -785

Usually not. Since RT-PCR is extremely sensitive, as little as 10–200 ng total RNA is sufficient for each 25–50 µl reaction mix, depending on the RT system. For abundant mRNA species, it is possible to use even less than 10 ng total RNA. For rare mRNA species, use a sequence-specific primer in the RT reaction to increase sensitivity. RNA content in various cells and tissues can be found here.

FAQ ID -111

No. UNG treatment does not provide any advantage for the QuantiFast and Rotor-Gene PCR kits because the mastermixes do not contain dUTP. Use the QuantiTect kits if you intend to use the UNG treatment.

FAQ ID -9092

No, please do not exchange Quantiscript Reverse Transcriptase of the QuantiTect Reverse Transcription Kit with QuantiTect RT Mix of the QuantiTect Probe RT-PCR Kit.

Although both are an optimized mixture of Omniscript and Sensiscript Reverse Transcriptases, the mixture provided in the QuantiTect Reverse Transription Kit is optimized for random priming in a two-step reaction, whereas the mixture in the QuantiTect Probe RT-PCR Kit is optimized for gene-specific priming in a one-step RT-PCR reaction.

FAQ ID -1066

Yes, you can substitute the RT Primer Mix supplied in the FastLane Cell cDNA Kit and the QuantiTect Reverse Transcription Kit with your gene-specific primers. We suggest optimizinig the primer concentration by titration, starting at 1 uM, and gradually decreasing it to 0.5 uM final concentration in the reaction. Optimal amounts will depend on the specific primers you are using.

FAQ ID -812

For best results, all RNA samples should be suspended in RNase-free water. Alternatively, RNase-free 1 mM sodium citrate (pH 6.5) or 10 mM Tris buffer (pH 7.0) may be used. Do not use DEPC-treated water, as most DEPC preparations are contaminated with molecules that are inhibitory to reverse transcription and/or PCR. For long-term storage, RNA preps may be stored at -70 ºC in RNase-free water, or the buffers listed above, or precipitated in ethanol or isopropanol. In order to avoid repeated freeze-thaw cycles, it is recommended that frozen RNA samples be stored as multiple, single-use aliquots.

FAQ ID -2659

If the extra peaks seem irregular or noisy, do not occur in all samples, and occur at temperatures less than 70 ºC, then these peaks may not represent real PCR products and instead may represent artifacts caused by instrument settings.

Usually extra peaks caused by secondary products are smooth and regular, occur reproducibly in most samples, and occur at temperatures greater than 70 ºC. Characterization of the product by agarose gel electrophoresis is the best way to distinguish between these cases. If only one band appears by agarose gel then the extra peaks in the dissociation curve are instrument artifacts and not real products. If this is the case, refer to the thermal cycler user manual, and confirm that all instrument settings (smooth factor, etc.) are set to their optimal values.

FAQ ID -90990

The REST 2009 (Relative Expression Software Tool) software applies mathematic models that compensate for the different PCR efficiencies of the gene of interest and reference genes. In addition, the software can use multiple reference gene normalization to improve the reliability of result, as well as provides statistical information suitable for robust comparison of expression in groups of treated and untreated. QIAGEN offers the REST 2009 software free of charge.

FAQ ID -9095

The FastLane Cell cDNA Kit and QuantiTect Reverse Transcription Kit contain a unique buffer, called gDNA Wipeout Buffer, which ensures complete removal of gDNA after a brief incubation step.

FAQ ID -783

The Ct or threshold cycle value is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal significantly above the background fluorescence. At the threshold cycle, a detectable amount of amplicon product has been generated during the early exponential phase of the reaction. The threshold cycle is inversely proportional to the original relative expression level of the gene of interest.

FAQ ID -2682

Check the template quality and integrity by amplifying an endogenous control gene. Check the amplicon by QIAxcel Advanced system or agarose gel electrophoresis to show that amplification was successful.

Determine whether the gene of interest is expressed in your sample. See How can I find out if my gene of interest is express in a specific tissue type or cell line. Ensure the assay setup and cycling conditions are correct, and that the data collection channel matches the emission wavelength of the fluorescent dye used. Use a control sample in which the gene of interest is definitely expressed.

If the issue persists, please send the original run file to QIAGEN Technical Services.

FAQ ID -9091

For duplex analysis, using non-fluorescent quenchers (e.g., Black Hole Quencher^®) is preferred over fluorescent quenchers (e.g., TAMRA fluorescent dye). For triplex and 4-plex analysis, QIAGEN strongly recommends using non-fluorescent quenchers. Generally, use the green channel, the yellow channel, and the orange and crimson channels to detect the least abundant target, the second least abundant target, and the two most abundant targets, respectively. For instrument-specific recommendations, please see the handbooks for the QuantiTect Multiplex PCR kit, QuantiFast Multiplex kit or Rotor-Gene Multiplex kit.

FAQ ID -9094

The gDNA wipeout buffer incubation step can be skipped when the total RNA is free from genomic DNA. However, the gDNA wipeout buffer is still required to be added because the reverse transcription step is optimized in the presence of components in the gDNA wipeout buffer.

FAQ ID -9098

Store the standards at a high concentration in aliquots at -20^oC to -70^oC. If using low concentrations, stabilize standards with carrier nucleic acid. It is always best to use freshly diluted standards for each experiment. If possible, use siliconized tubes for standard (and target) dilutions. This will prevent any unspecific binding of nucleic acids to the plastic.

FAQ ID -9099

Yes, reactions using the QuantiTect Reverse Transcription Kit can be scaled up. Please scale up all reaction components proportionally.

FAQ ID -1063

Reliable HRM analysis results depend on template quality, highly specific HRM PCR kit with a saturation dye, a real-time instrument with HRM capability, and powerful software package. Factors critical for successful HRM analysis are:

Use the same genomic DNA purification procedure for all samples being analyzed by HRM. This avoids variation due to differing composition of elution buffers.
DNA template concentrations should be normalized using the same dilution buffer. Ensure the C_T values are below 30 and differ no more than 3 C_T values across individual samples.
Design assays with amplicon length 70–350 bp. For SNP analysis, use amplicon length 70–150 bp.
Always start with 0.7 µM primer concentration

For more details, please refer to the HRM Technology – FAQs and the Critical Success Factor for HRM performance.

FAQ ID -9097

No, QuantiTect Primer Assays are supplied as lyophilized, premixed primer pairs. Reaction components for SYBR Green real-time RT-PCR must be purchased separately.

For two-step RT-PCR, the QuantiTect Reverse Transcription Kit and QuantiTect SYBR Green PCR Kit are recommended
For one-step RT-PCR, the QuantiTect SYBR Green RT-PCR Kit is recommended

To find a QuantiTect Primer Assay for your target gene of interest, please visit our GeneGlobe data base.

FAQ ID -1143

Shorter amplification products facilitate high PCR efficiencies. Ideally, amplicon length should be less than 150 bp for optimal amplification efficiency. PCR efficiencies close to 100% are a crucial prerequisite for accurate quantification of target copy numbers in real-time PCR.

FAQ ID-751

The QuantiTect Reverse Transription Kit should be stored at -20°C immediately upon receipt. We recommend to aliquot the RT Primer Mix and keep it at -20°C.

If the kit is being used routinely, it may be convenient to prepare a premix of RT Primer Mix and 5x Quantiscript RT Buffer at a ratio of 1:4. Aliquot the premix and store at -20°C.

FAQ ID -1077

No, the T-Script® enzyme of the QuantiTect Whole Transcriptome Kit is an optimized blend for whole transcriptome amplification and cannot be substituted by Quantiscript Reverse Transcriptase of the QuantiTect Reverse Transcription Kit, or any other reverse-transcription enzyme.

FAQ ID -1617

Depending on primer design and copy number of target, primer-dimer may occur and its signal might be detected. Typical strategies against this are to optimize PCR conditions and/or redesign the assay.

Alternatively, an additional data-acquisition step can be added to the 3-step cycling protocol. First, determine the melting temperatures (T_m) for both the amplicon and the primer-dimer. Then, add a 15 second data-acquisition step with a temperature that is higher than the primer-dimer T_m, but approximately 3ºC lower than the specific amplicon T_m.

FAQ ID -9096