For fast cDNA synthesis enabling sensitive real-time two-step RT-PCR for gene expression analysis
cDNA synthesis and gDNA removal in only 20 minutes
High cDNA yields even from low-abundance transcripts
cDNA synthesis from 5' and 3' regions of transcripts
No need to design RNA-specific primers or probes
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.
For 200 x 20 µl reactions: 4 x 100 µl 7x gDNA Wipeout Buffer, 4 x 50 µl Quantiscript Reverse Transcriptase, 4 x 200 µl 5x Quantiscript RT Buffer, 4 x 50 µl RT Primer Mix, 4 x 1.9 ml RNase-Free Water
For 400 x 20 µl reactions: 800 µl 7x gDNA Wipeout Buffer, 400 µl Quantiscript Reverse Transcriptase, 1.6 ml 5x Quantiscript RT Buffer, 400 µl RT Primer Mix, 8 x 1.9 ml RNase-Free Water
The QuantiTect Reverse Transcription Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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.
Effective genomic DNA removal for accurate real-time RT-PCR.
Real-time, two-step RT-PCR analysis of β-actin with (+RT) or without (-RT) reverse transcriptase. cDNA was synthesized from 100 ng total RNA, and real-time PCR was performed in duplicate on the LightCycler 2.0 using the QuantiTect SYBR® Green PCR Kit. The β-actin-specific primers detected both mRNA and genomic DNA sequences. Control reactions with no template were also performed (green). [A] The RT step was carried out using the QuantiTect Reverse Transcription Kit. The red, flat –RT plot indicates efficient removal of residual genomic DNA. [B] The RT step was carried out using a kit from Supplier I (Enzyme SIII). The purple –RT plot indicates amplification of residual genomic DNA.
Sensitive detection of a target at the 5' region of a 12.5 kb transcript.
Real-time, two-step RT-PCR of a target located at the 5' region of the mouse dystrophin gene (about 12.5 kb upstream of the poly-A site). Total RNA purified from mouse testis was reverse transcribed with the QuantiTect Reverse Transcription Kit as well as with reverse transcriptases from Supplier I and Supplier R. Identical volumes of triplicate reverse-transcription reactions were analyzed by real-time PCR on the LightCycler system. The error bars show the standard deviation for each set of triplicates. Compared with the other two kits, the QuantiTect Reverse Transcription Kit generated much higher amounts of cDNA (indicated by the lower CT values) and provided greater reproducibility in real-time RT-PCR (indicated by the smaller error bars). RFU: relative fluorescence units.
(Data kindly provided by Dr. Andrej-Nikolai Spiess, Department of Molecular Andrology, University Hospital Hamburg, Germany).
Higher sensitivity in real-time, two-step RT-PCR.
Real-time, two-step RT-PCR analysis of [A] TGFB2 (low expression) and [B] IL8 (higher expression). Total RNA was purified from human whole blood using the PAXgene Blood RNA system. cDNA was then synthesized from 1 µg RNA using the QuantiTect Reverse Transcription Kit, a kit from Supplier AII, or a kit from Supplier I. Real-time PCR was performed in duplicate on the ABI PRISM 7900 using the QuantiTect Probe PCR Kit and QuantiTect Gene Expression Assays for TGFB2 or IL8. The CT values for TGFB2 were lowest with the QuantiTect Reverse Transcription Kit, demonstrating that even low-abundance genes can be efficiently reverse transcribed and sensitively detected in real-time PCR.
Performance
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
CT values for IL12A
(low expression)
CT values for IL1RN
(higher expression)
Input RNA (ng)
QIAGEN
Supplier AII
QIAGEN
Supplier AII
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
Real-time, two-step RT-PCR analysis of IL12A and IL1RN using different amounts of input RNA. Total RNA was reverse transcribed using the QuantiTect Reverse Transcription Kit or a kit from Supplier AII. The cDNA synthesized was analyzed on the ABI PRISM 7900 using the QuantiTect Probe PCR Kit and QuantiTect Gene Expression Assays for IL12A or IL1RN. CT values were lower with the QIAGEN kit, especially for the medium-abundance IL12A gene (bold), indicating higher cDNA yields. N.D.: Not detected.
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.
Principle
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
Procedure
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.
Applications
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.
Features
Specifications
Applications
Quantification of (even low-abundance) transcripts
1 x 25 ml HotStarTaq Master Mix (contains 2500 units HotStarTaq DNA Polymerase PCR Buffer with 3 mM MgCl2, and 400 µM of each dNTP) and 1 x 50 ml RNase-Free Water
