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TRANSRTRO

Roche

Transcriptor Reverse Transcriptase

Quality Level

manufacturer/tradename

Roche

packaging

pkg of 200 reactions (03531287001)
pkg of 25 reactions (03531317001)
pkg of 50 reactions (03531295001)

application(s)

RT-qPCR: suitable

detection method

probe-based

General description

In retroviruses like the human immunodeficiency virus type 1 (HIV-1), reverse transcriptase (RT) is the core enzyme. HIV-1 RT is made of two subunits of 66 kDa and 51 kDa (p66 and p5l). A human endogenous retrovirus of the HERV-K family codes for reverse transcriptase (RT) enzyme.
A recombinant reverse transcriptase for the robust transcription of RNA fragments up to 14 kb used in two-step RT-PCR on thermal cyclers and real-time PCR instruments.

Application

Transcriptor Reverse Transcriptase is designed to transcribe RNA (mRNA, total RNA, viral RNA, and in vitro-transcribed RNA) from a variety of sources, using conventional thermal cyclers and real-time PCR instruments (e.g., the LightCycler® Instruments) for the following applications:

  • Synthesis of first-strand cDNA for use in subsequent amplification reactions
  • RT-PCR of GC-rich RNA templates
  • Cy3, Cy5, DIG, biotin, and aminoallyl labeling during cDNA synthesis
  • Retrieving and cloning the 5′ and 3′ termini of mRNA by RACE
  • Generation of cDNA libraries with large inserts
  • Dideoxy DNA sequencing
  • RNA sequencing
  • 3′-end labeling of DNA fragments
  • Generation of single-stranded probes for genomic footprints
  • In the reverse transcription of RNA from human Papillomavirus E6, cortical and striatal tissues, muscle biopsies of Becker muscular dystrophy samples and miRNA stem-loop from oocytes and human cerebral microvascular endothelial cells (hCMVECs)

Biochem/physiol Actions

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is essential for the catalytic conversion of single-stranded viral RNA into the double-stranded linear DNA that is integrated into host cell chromosomes.

Features and Benefits

  • Achieve high sensitivity in two-step RT-PCR.
Transcriptor Reverse Transcriptase is used in conventional thermal cyclers and real-time PCR instruments (e.g., the LightCycler®Instruments).
  • Obtain more full-length transcripts - up to 14 kb.
cDNA libraries with large inserts can be generated.
  • Reverse transcribe difficult templates.
The enzyme works well at elevated temperatures, thereby overcoming RNA secondary structure (e.g., GC-rich RNA templates) and facilitating optimal reaction conditions.
  • Efficiently label cDNA.
Cy3-, Cy5-, DIG-, biotin-, or aminoallyl-labeled nucleotides are incorporated during cDNA synthesis.

Components

  • Transcriptor Reverse Transcriptase, in storage buffer
  • Transcriptor RT buffer, 5x concentrated

Quality

Each lot of Transcriptor Reverse Transcriptase is routinely function tested in RT-PCR:

  • with a conventional thermal cycler to detect a 10-kb fragment from the human dystrophin gene, starting from human skeletal muscle total RNA
  • with the LightCycler® Instrument to detect 5 x 102 to 5 x 106 copies of in vitro-transcribed human PBGD RNA. The results are defined in fixed crossing points and fixed fluorescence intensity.

Preparation Note

Volume activity: 20 U/μl.
Transcription of long, rare, or difficult RNA targets
Transcriptor Reverse Transcriptase is recommended for RT-PCR of:

  • long targets, because it can transcribe up to 14 kb RNA templates
  • rare targets, because it has high sensitivity
  • GC-rich targets, because it can operate at high temperatures (up to +65oC) to eliminate problems associated with extensive secondary structure

Labeling for many applications
Transcriptor Reverse Transcriptase is also recommended for preparing labeled cDNA, since it accepts a wide variety of modified nucleotides (including Cy3-, Cy5-, DIG-, Biotin-, or aminoallyl-labeled dNTPs).
Reaction requirements
Transcriptor accepts ssRNA and ssDNA templates and requires a primer for transcription.

Other Notes

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

Legal Information

LightCycler is a registered trademark of Roche

RIDADR

NONH for all modes of transport

WGK Germany

WGK 1

Flash Point(F)

does not flash

Flash Point(C)

does not flash

Certificate of Analysis

Certificate of Origin

Identification of an active reverse transcriptase enzyme encoded by a human endogenous HERV-K retrovirus.
Berkhout B, et al.
Journal of Virology, 73(3), 2365-2375 (1999)
Valdimara C Vieira et al.
mBio, 5(6) (2014-12-30)
Several recent studies have converged upon the innate immune DNA cytosine deaminase APOBEC3B (A3B) as a significant source of genomic uracil lesions and mutagenesis in multiple human cancers, including those of the breast, head/neck, cervix, bladder, lung, ovary, and other...
Tracking disease progression non-invasively in Duchenne and Becker muscular dystrophies
Spitali P, et al.
Journal of Cachexia, Sarcopenia and Muscle, 9, 715-726 (2018)
Pietro Spitali et al.
Journal of cachexia, sarcopenia and muscle, 9(4), 715-726 (2018-04-24)
Analysis of muscle biopsies allowed to characterize the pathophysiological changes of Duchenne and Becker muscular dystrophies (D/BMD) leading to the clinical phenotype. Muscle tissue is often investigated during interventional dose finding studies to show in situ proof of concept and...
Expression profiles and function analysis of microRNAs in postovulatory aging mouse oocytes
Wang TY, et al.
Aging, 9, 1186-1186 (2017)

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