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RT-PCR in one step
Single-tube RT-PCR systems for convenient, sensitive and reproducible RT-PCR amplification of a specific target RNA RobusT™ I RT-PCR Kit
RobusT™ II RT-PCR Kit
Description
RT-PCR, coupling reverse transcription (RT) of mRNA with PCR amplification of the resulting cDNA, is a rapid and sensitive method for analyzing gene expression and producing cDNA for cloning. RobusT™ RT-PCR Kits are designed to improve the sensitivity and efficiency of cDNA synthesis and PCR amplification. The reactions are designed for a convenient one-tube- one-buffer system. A unique reverse transcriptase/DNA polymerase combination in two different kit formats guarantees the excellent features of the kits.
The Robus™ T I Kit is based on the highly thermostable AMV-RT, capable of producing cDNA at elevated temperatures and thus minimizing the problems originating from secondary RNA structures. The RobusT™ II Kit is based on the sensitive properties of M-MuLV RNase H- facilitating the amplification of minor amounts of starting mRNA material. The excellent performance of the two systems is then built into DyNAzyme™ EXT DNA Polymerase, which has proved to be a powerful amplification enzyme capable of high fidelity DNA synthesis with high yields.
RobusT RT-PCR Kits perform cDNA synthesis and PCR amplification of cDNA successively in a single tube during a continuous thermal cycling. Use of the same buffer conditions and site-specific primers eliminates the need to open the reaction tube and adjust conditions between the RT and PCR steps. In addition to increased sensitivity, the one-tube system minimizes the risk of contamination and the requirement for hands-on time. One-step RT-PCR is easier to use when processing large numbers of samples.
Finnzymes offers also tools for two-step RT-PCR: AMV Reverse transcriptase, M-MuLV RNase H- Reverse Transcriptase and DyNAzyme EXT DNA Polymerase are available as individual enzymes. These enzymes are supplied with optimized 10x buffers. Other reaction components for two-step process (e.g. dNTP's) are also available. Stand alone enzymes are often used when the same cDNA source is used for analysing several different genes. The advantage of uncoupled RT-PCR is that multiple transcripts can be analyzed from a single RT-reaction. The sample can also be stored more safely as cDNA than as RNA.
Applications
- Robust, high-yield RNA amplification
- Generation of cDNA products with high fidelity for cloning, sequencing and microarray purposes
- Quantification of gene expression and reliable expression profiling
- Clinical diagnostics: detection and quantification of infectious microorganisms, cancer cells and genetic disorders
Advantages
- Capable of amplifying long targets, up to 6.0 kb
- One-tube system allows rapid, sensitive and reproducible analysis of RNA with minimal risk of sample contamination
- Amplifies products from a wide variety of total RNA or mRNA sources
