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PyroMark OneStep RT-PCR Kit

For highly sensitive and specific one step RT-PCR, optimized for Pyrosequencing analysis

Features

  • Dedicated end-point RT-PCR solution for Pyrosequencing analysis
  • Efficient reverse transcription and subsequent amplification in one tube
  • Unique enzyme mix and buffer for high specificity and sensitivity
  • Streamlined protocols eliminate the need for optimization
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PyroMark OneStep RT-PCR Kit (200)

Cat. No. / ID: 978803

For 200 reactions: QIAGEN OneStep RT-PCR Enzyme Mix, optimized QIAGEN OneStep RT-PCR Buffer, 10x CoralLoad Concentrate, 5x Q-Solution, dNTP Mix, and RNase-Free Water
Reactions
200
50
The PyroMark OneStep RT-PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Product Details

The PyroMark OneStep RT-PCR Kit is specifically optimized for Pyrosequencing analysis, enabling efficient reverse transcription of the RNA template, and subsequent highly specific and reliable amplification of the cDNA. The entire process takes place in one tube, affording speed and ease of use and reducing the risk of contamination.

Principle

The PyroMark OneStep RT-PCR Kit is the first RT-PCR kit that is dedicated and optimized for preparing samples for subsequent Pyrosequencing analysis. The kit is designed for simple but sensitive one-step RT-PCR using any RNA template. The RT-PCR product generated can be used for a variety of Pyrosequencing applications, such as virus detection and genotyping, allele-specific gene expression analysis, and detection of mRNA splicing isoforms. A unique enzyme combination and specially developed reaction buffer ensure efficient, highly specific reverse transcription and PCR in one tube, without the need for optimization. Q-Solution is included in the kit to enable efficient amplification of templates with complex secondary structures and high GC content, while CoralLoad Concentrate ensures easy loading and visualization of the RT-PCR product on agarose gels.

QIAGEN OneStep RT-PCR Enzyme Mix

The QIAGEN OneStep RT-PCR Enzyme Mix contains a specially formulated enzyme blend, specifically formulated for both reverse transcription and PCR amplification.

  • Omniscript and Sensiscript Reverse Transcriptases provide highly efficient and specific reverse transcription. Both reverse transcriptases exhibit a higher affinity for RNA, facilitating transcription through secondary structures that would normally inhibit other reverse transcriptases. Omniscript Reverse Transcriptase is specially designed for reverse transcription of RNA amounts greater than 50 ng, and Sensiscript Reverse Transcriptase is optimized for use with very small amounts of RNA (<50 ng). This special enzyme combination provides highly efficient and sensitive reverse transcription from 1 pg to 2 μg of starting RNA.
  • HotStarTaq DNA Polymerase provides hot-start PCR for highly specific amplification. The chemically modified HotStarTaq DNA Polymerase is completely inactive during the reverse transcription step, and does not interfere with the reverse transcriptase process. After reverse transcription, reactions are heated to 95°C for 15 min to activate HotStarTaq DNA Polymerase and to simultaneously inactivate the reverse transcriptases. This hot-start procedure using HotStarTaq DNA Polymerase eliminates extension from nonspecifically annealed primers and primer dimers in the first PCR cycle, ensuring highly specific and reproducible PCR.
QIAGEN OneStep RT-PCR Buffer

QIAGEN OneStep RT-PCR Buffer is designed to enable both efficient reverse transcription and specific amplification. The unique buffer composition allows reverse transcription to be carried out at a high temperature (50°C), improving the efficiency of the reaction by disrupting secondary structures, which is particularly important when using limiting amounts of template RNA. The combination of QIAGEN enzymes and the unique formulation of the buffer also ensures high PCR efficiency in one step RT-PCR. This buffer ensures specific primer annealing over a wider range of annealing temperatures and Mg2+ concentrations than conventional PCR buffers. This minimizes the need for optimization of RT-PCR by varying the annealing temperature or the Mg2+ (see figure " Increased specificity of primer annealing").

CoralLoad Concentrate

The PyroMark OneStep RT-PCR Kit is also supplied with CoralLoad Concentrate, which contains a gel loading reagent and two marker dyes — an orange and a red dye (see figure " CoralLoad Concentrate"). RT-PCR products amplified in the presence of CoralLoad Concentrate can be loaded directly onto an agarose gel.

Q-Solution

The PyroMark OneStep RT-PCR Kit includes Q-Solution, an innovative additive that modifies the melting behavior of nucleic acids. Q-Solution facilitates reverse transcription and amplification of templates with a high GC content or a high degree of secondary structure.

See figures

Procedure

Reverse transcription and PCR are carried out sequentially in the same tube. All components required for both reactions are added during reaction setup, so there is no need to add additional components once the reaction has started. The optimized cycling conditions include a 30 minute reverse transcription reaction, followed by activation of the HotStarTaq DNA Polymerase and subsequent template amplification.

Applications

The PyroMark OneStep RT-PCR Kit is specially optimized for subsequent Pyrosequencing applications, such as:

  • Virus detection
  • Virus genotyping
  • Allele-specific gene expression analysis
  • Detection of mRNA splicing isoforms

Supporting data and figures

Resources

Safety Data Sheets (1)
Download Safety Data Sheets for QIAGEN product components.
Brochures & Guides (1)
Kit Handbooks (1)

FAQ

Where can I order the Streptavidin Sepharose beads for pyrosequencing?
The recommended Streptavidin Sepharose High Performance beads for pyrosequencing can be ordered at GE healthcare with the catalog no 17-5113-01.

The PyroMark Q48 Autoprep protocol uses magnetic streptavidin-coated Sepharose® beads (PyroMark Q48 Magnetic Beads), which bind to the biotinylated PCR strand.

PyroMark Q48 Magnetic Beads can be ordered at QIAGEN with the catalog no 974203.

FAQ ID -2850
How many nucleotides of a homopolymer can be resolved in pyrosequencing?
In the range of 3-5 bases can be resolved depending on the sequence context and base. If it is possible sequencing of a homopolymer of more than 3-5 nucleotides should be avoided by resetting the sequencing primer.
FAQ ID -2871
What is the reason for a high substrate peak in the pyrosequencing pyrogram?
Usually pyrophosphate or dATP/ATP contamination in the sample or in the buffer can cause a high substrate peak. Large amounts of pyrophosphate are generated in the PCR reaction and might be carried over to the sequencing reaction. Check the PyroMark buffers and reagents and use new ones.
FAQ ID -2879
Does the Pyromark Assay Design or application software give any cycling conditions for individual assay primers or a PCR setup pipetting scheme?
The PyroMark Assay Design and application software do not support PCR setup with a pipetting scheme or PCR cycling conditions. General recommendations how to setup and optimization of the PCR reaction are contained in the PyroMark PCR handbook.
FAQ ID -2862
How do I reduce background peaks in the pyrosequencing pyrogram?
There are several reasons for a high assay background; the template can form secondary structures which are extended or the primers itself form dimmers which serve as template. Perform accurate sequencing controls (e.g. PCR or sequencing primer only) as recommended in the PyroMark User Manual to observe this kind of background. In addition, an unspecific priming of primer to template or unspecific annealing of sequencing primer to template might also be a background cause. Please check your complete primer design and if needed, perform a redesign. Try to lower the primer concentration as possible to avoid excess of primer.
FAQ ID -2877
How many times can vacuum troughs be re-used with the PyroMark Vacuum Preparation Stations?
There is no precise recommendation how many times these troughs on the PyroMark Vacuum Preparation Stations (Q24 and Q96) can be re-used. It depends on the individual handling and cleaning (with water).
FAQ ID -2848
Which purity grade is recommended for pyrosequencing primers?
Only the biotinylated primer needs to be HPLC purified whereas the other primers require standard desalting only.  Pyrosequencing primers can be ordered here.
FAQ ID -2832
What is the reason for split peaks appearing in between dispensations on my pyrosequencing pyrogram?
The PyroMark cartridge needle can be blocked or damaged. Clean the cartridge or exchange with a new one. Check for correct reagent cartridge and cartridge method used in the run. Check if the reagent cartridge cover was closed properly. Make sure that the cartridge was dry after cleaning because nucleotide droplets might be caught at the needle tip and fall down at any time. or exchanged.
FAQ ID -2881
What is the reason for signals ceasing in the middle of a pyrosequencing run?
The cartridge needle can be blocked or damaged causing a dispensation error. Clean the cartridge following the guidelines or repeat the run with a new cartridge. On the other hand if high amounts of template have been used resulting in very high signals (>100 RLU), the substrate for the sequencing reaction might be depleted. In this case template conditions should be optimized.
FAQ ID -2875
Can unused wells in a pyrosequencing plate be used in the next run?
In principle it’s possible to use so far unused pyrosequencing wells for the next run and leave the already used wells empty. However, due to contamination risk when cleaning and handling plates QIAGEN does not recommend this.
FAQ ID -2872
How do I prevent a drifting baseline in my pyrosequencing pyrogram?
Let the PyroMark instrument warm up (about 60 minutes) to adapt to room temperature before use. Make sure the ambient room temperature is within range 18-28°C.
FAQ ID -2878