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TOSRTRO

Roche

Transcriptor One-Step RT-PCR Kit

Quality Level

mfr. no.

Roche

packaging

pkg of 150 reactions (04655885001)
pkg of 50 reactions (04655877001)

General description

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.

Application

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)
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.

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.

Components

  • 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.

Quality

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

For life science research only. Not for use in diagnostic procedures.

Hazard Statements

Precautionary Statements

RIDADR

NONH for all modes of transport

Flash Point(F)

does not flash

Flash Point(C)

does not flash

Certificate of Analysis
Certificate of Origin
Georg von Massow et al.
Infection and drug resistance, 12, 947-955 (2019-05-24)
Hepatitis C virus (HCV) is a highly variable infectious agent, classified into 8 genotypes and 86 subtypes. Our laboratory has implemented an in-house developed high-resolution HCV subtyping method based on next-generation sequencing (NGS) for error-free classification of the virus using...
Detection of murine leukemia virus or mouse DNA in commercial RT-PCR reagents and human DNAs
<BIG><BIG>Zheng HQ, et al.</BIG></BIG>
Testing, 6 (2011)
Whole-genome characterization and resistance-associated substitutions in a new HCV genotype 1 subtype
<BIG><BIG>von MG, et al.</BIG></BIG>
Infection and Drug Resistance, 12, 947-947 (2019)
Claire Harris et al.
mAbs, 11(8), 1452-1463 (2019-10-02)
Protein primary structure is a potential critical quality attribute for biotherapeutics. Identifying and characterizing any sequence variants present is essential for product development. A sequence variant ~11 kDa larger than the expected IgG mass was observed by size-exclusion chromatography and...
HaoQiang Zheng et al.
PloS one, 6(12), e29050-e29050 (2011-12-30)
The xenotropic murine leukemia virus (MLV)-related viruses (XMRV) have been reported in persons with prostate cancer, chronic fatigue syndrome, and less frequently in blood donors. Polytropic MLVs have also been described in persons with CFS and blood donors. However, many...
Protocols
Coronavirus qPCR Primer and probe sets for the detection of SARS-CoV-2 (Corona Virus), with additional real-time RT-PCR, RT-qPCR and supporting reagents available.
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