PyroMark OneStep RT-PCR Kit

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.

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.

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.

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