General description

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.

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.

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 +65^oC) 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

usage: sufficient for 25 . reactions, sufficient for 50 . reactions, sufficient for 500 . reactions. Quality Level: 100. feature: dNTPs included: no, hotstart: no. manufacturer/tradename: Roche. packaging: pkg of 200 reactions (03531287001), pkg of 25 reactions (03531317001), pkg of 50 reactions (03531295001). technique(s): RT-PCR: suitable, RT-qPCR: suitable. detection method: probe-based. Storage Class Code: 12 - Non Combustible Liquids. WGK: WGK 1. Flash Point(F): does not flash. Flash Point(C): does not flash.