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.
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).
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.
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.