MasterAmp™ RT-PCR Kit

* Covered by issued and pending patents

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Last modified: 2008-07-24 11:43:49

The MasterAmp™ RT-PCR Kit for High Sensitivity is a single-enzyme, single-tube RT-PCR system. It is capable of amplifying RNA from a single gene in as little as 1pg of total cellular placental RNA (e.g., human chorionic gonadotropin Figure 1). It is ideal for amplifying low-copy-number RNA transcripts.

The MasterAmp™ RT-PCR Kit for High Sensitivity uses RetroAmp™ RT DNA Polymerase, a thermostable enzyme that has reverse transcriptase activity at ≥60°C and lacks RNase H activity. Reverse transcription at higher temperatures reduces RNA secondary structure and increases the specificity and sensitivity of cDNA synthesis. The sensitivity of this kit is further augmented because both RT and PCR occur in a single tube. All cDNA synthesized during RT is used as a template for PCR amplification.

The kit also incorporates MasterAmp™ PCR Enhancer (with betaine)*, which further improves the specificity, consistency, and sensitivity of RT-PCR (Figure 2)by reducing DNA and RNA secondary structure, eliminating sequence composition dependence of nucleic acid melting, and reducing pauses during DNA synthesis.

Figure 1. RT-PCR amplification of human chorionic gonadotropin-alpha (hCGα) from decreasing amounts of placental total cellular RNA using MasterAmp™ RT-PCR Kit for High Sensitivity. RNA was isolated using the MasterPure™ Complete DNA and RNA Purification Kit. A 40-cycle amplification was performed using decreasing amounts of total RNA following the protocol supplied.

Figure 2. Human CGα was amplified from various amounts of total human placental RNA using MasterAmp™ RT-PCR Kit for High Sensitivity or kits from other manufacturers. All experiments were carried out strictly according to each manufacturer's protocols.

Protocols for One-Step and Two-Step Applications

One-Tube One-Step One-Enzyme RT-PCR for Gene Specific Primers Amplify a specific RNA by performing first-strand cDNA synthesis and PCR in one tube using gene-specific primers.
One-Tube Two-Step RT-PCR Using Oligo(dT) or Random Primers for 1st-Strand cDNA Synthesis, Optionally Followed by Gene-Specific Primers for PCR Perform first-strand cDNA synthesis using random oligo(dT) primers. In the subsequent PCR reaction, use DNA-generated and specific primers.

Last modified: 2008-05-14 16:46:46

Applications

• Detection of RNA in low copy number
• All RT-PCR applications where sensitivity is of primary concern
• Quantitative RT-PCR
• Multiplex RT-PCR

Benefits

• High sensitivity RT-PCR from as little as 1 pg of total cellular RNA
• Out-performs similar products from other vendors
• Reduces effect of RNA secondary structure by performing RT reactions at higher temperature
• MasterAmp PCR Enhancement Technology enhances PCR performance with GC-rich and other difficult templates

Last modified: 2008-05-14 16:46:46

Protocols for: MasterAmp™ RT-PCR Kit for High Sensitivity

(catalogue number RT71225 / RT712100)

Please note: all protocols off site are the responsibility of the products supplier

Last modified: 2008-05-14 16:46:46