Description
General description
For hot start one-step RT-PCR, utilizing the enzyme mixture of Transcriptor Reverse Transcriptase, Protector RNase Inhibitor, and the Expand System, with an innovative hot start buffer (patents pending).
One-step RT-PCR is a very sensitive technique for determining the presence or absence of RNA templates, or to quantify the levels of expression through qualitative, semi-quantitative, or quantitative analysis of RNA transcription levels (e.g., for virus-level quantification). This RT-PCR method also facilitates the amplification of rare messages for cloning. The one-step format is convenient, shows reduced reaction-to-reaction variability, and prevents contamination by minimizing hands-on steps.
The Transcriptor One-Step RT-PCR Kit offers the outstanding benefits of Transcriptor Reverse Transcriptase, as well as the advantages of a hot start one-step RT-PCR format.
The Transcriptor One-Step RT-PCR Kit provides all components required for one-step RT-PCR (except primers and template) in a convenient format. The 50-reaction pack size also includes 10 control reactions (control RNA and control primer mix).
The kit′s RT-PCR Enzyme Mix contains four different enzymes: Transcriptor Reverse Transcriptase ensures sensitive and robust reverse transcription with high yield; Protector RNase Inhibitor, fully active at elevated temperatures, provides maximum template protection during reverse transcription; and the Expand System – a blend consisting of Taq DNA Polymerase and a proofreading polymerase – minimizes the possibility of mutations, offering high yield and fidelity in the PCR.
The optimized RT-PCR Reaction Buffer includes high quality dNTPs and provides the overall improved performance of a hot start system. The buffer′s unique hot start component binds and sequesters primers at lower temperatures to prevent the primers from binding to nonspecific sites. Another component binds magnesium, in a temperature-dependent manner, to prevent uncontrolled DNA synthesis. This new buffer formulation is effective during reverse transcription as well as PCR, resulting in increased specificity and sensitivity.
The combination of optimized reaction buffer and enzyme mix ensures efficient transcription of difficult templates with high secondary structure and GC content without increasing reaction temperature.
Application
The Transcriptor One-Step RT-PCR Kit is designed for fast, sensitive, and specific end-point RT-PCR analysis of RNA (total RNA, mRNA, in vitro-transcribed RNA, or viral RNA) using gene-specific primers.
Featuring an innovative reaction buffer, the kit combines sensitive, high-yield reverse transcription with the improved performance of a hot start system for high-fidelity, high-yield amplification of a variety of templates, including GC-rich RNA.
The kit offers a convenient, highly sensitive one-step RT-PCR method to generate transcripts up to 6.5 kb for applications such as:
Qualitative, semi-quantitative, or quantitative analysis of RNA transcription levels
Determining the presence or absence of RNA templates
Quantifying gene expression through RNA analysis (e.g., for virus-level quantification)
Cloning of RNA up to 6.5 kb
The one-step format is convenient, reduces reaction-to-reaction variability, and prevents contamination by minimizing hands-on steps.
Transcriptor One-Step RT-PCR Kit has been used in the amplification of:
hepatitis C virus (HCV) fragments
heavy chain CH3 region and light chain polyA region
murine leukemia virus (MLV)
Features and Benefits
Obtain high sensitivity and yield in a wide amplification range:
Detect as little as 1 fg of total RNA.
Achieve high specificity and reduce primer-dimers
Count on excellent performance with the kit′s proprietary hot start buffer (patents pending).
Produce long fragments:
Generate transcripts up to 6.5 kb.
Transcribe a variety of templates, even the most difficult (e.g., GC-rich RNA):
Overcome high secondary structure with thermostableTranscriptor Reverse Transcriptase in combination with an optimized buffer.
Save time and improve results with a convenient kit:
Minimize pipetting steps and prevent contamination by using a premixed enzyme blend and optimized buffer that includes PCR-grade dNTPs and hot start components.
Protect your RNA sample:
Safeguard your RNA from degradation by RNases with the supplied thermostable Protector RNase Inhibitor that is functional during one-step RT-PCR when other RNase inhibitors fail.
Increase confidence in your results:
Use the supplied control reactions to monitor performance.
Analysis Note
Each lot of the Transcriptor One-Step RT-PCR Kit is function tested in RT-PCR. RT-PCR is performed with 1000 copies of HAV RNA, in vitro transcript (also provided with Cat.No 04 655 877 001). The control reaction setup and the subsequent RT-PCR with primers for a 246 bp HAV RNA fragment are performed using the standard RT-PCR protocol (reverse transcription at +50°C for 30 minutes) provided in the package insert. With 1,000 copies of target a clearly visible band is obtained after agarose gel electrophoresis and ethidium bromide staining.
In addition, RT-PCR is performed on a dilution series of human liver total RNA (10 ng, 1 ng, and 0.1 ng). RT-PCR with specific primers for a 2.5 kb fragment of ApoB is performed using the standard RT-PCR protocol (reverse transcription at +50°C for 30 minutes) provided in the package insert. With 1 ng of RNA a clearly visible band is obtained after agarose gel electrophoresis and ethidium bromide staining.
Other Notes
Reverse Transcriptases Overview
RT-PCR Enzyme Mix containing Transcriptor Reverse Transcriptase, Protector RNase Inhibitor, and the Expand System
RT-PCR Reaction Buffer containing hot start mediating components and dNTPs – 5x concentrated
Water, PCR Grade
Control RNA – HAV RNA, in vitro transcript *
Control primer mix HAV RNA (forward and reverse primer) *
* Included only in the 50-reaction pack size.
For life science research only. Not for use in diagnostic procedures.
