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Transcriptor Reverse Transcriptase

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.

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

Product No. 03531317001
Pack Size 25 reactions

This link will direct you outside of Roche to our exclusive global distribution partner's website.

Product No. 03531295001
Pack Size 50 reactions

This link will direct you outside of Roche to our exclusive global distribution partner's website.

Product No. 03531287001
Pack Size 200 reactions

This link will direct you outside of Roche to our exclusive global distribution partner's website.

  • 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.

 

Contents

  1. Transcriptor Reverse Transcriptase, in storage buffer
  2. Transcriptor RT buffer, 5x concentrated

 

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

Figure 1: Detection of mouse β-actin mRNA by two-step RT-PCR on a conventional thermal cycler.
Various amounts of mouse liver total RNA were reverse transcribed with Transcriptor Reverse Transcriptase by oligo (dT)15 priming in a 20-μl reaction. Five microliters of the reverse transcription reaction then served as a template for the FastStart High Fidelity PCR System in a PCR to amplify a 324-bp fragment of the mouse β-actin gene. All reactions were performed in duplicate.



Figure 2: Sensitivity in one-step RT-PCR of a 2-kb dystrophin RNA fragment (comparison of Transcriptor to other reverse transcriptases), with Expand High Fidelity Enzyme Blend used as the PCR component.
Transcriptor Reverse Transcriptase exhibited the highest sensitivity.



Figure 3: Overcome difficult RNA templates by using Transcriptor Reverse Transcriptase at up to +65°C. Transcriptor Reverse Transcriptase or other reverse transcriptases were used with a specific primer to reverse transcribe a 4.9-kb fragment (GC content: 70%) of human 28S ribosomal RNA at +55°C, +60°C, and +65°C.
During the two-step RT-PCR, FastStart Taq DNA Polymerase and its associated GC-RICH Resolution Solution were used to amplify a 389-bp fragment on a conventional thermal cycler. Due to its high thermostability, Transcriptor Reverse Transcriptase can be used at up to +65°C, making it, in combination with FastStart Taq DNA Polymerase, the product of choice when GC-rich templates must be reverse transcribed and amplified in a subsequent PCR.
Lane 1: Transcriptor Reverse Transcriptase
Lane 2: RNase HM-MuLV from Supplier A
Lane 3: Avian RNase H- transcriptase from Supplier A




Figure 4: Produce full-length transcripts up to 14 kb for sensitive, high-yielding RT-PCR with Transcriptor Reverse Transcriptase. See Figure 5 for details.



Figure 5: Produce full-length transcripts up to 14 kb for sensitive, high-yielding RT-PCR with Transcriptor Reverse Transcriptase.
Various amounts of human skeletal muscle total RNA were reverse transcribed with Transcriptor Reverse Transcriptase and other reverse transcriptases via oligo (dT) priming in a 20-μl reaction, producing a 14-kb mRNA for the human dystrophin gene, one of the longest eukaryotic mRNAs.
In a subsequent PCR (Figure 4), 2 μl of the RT reaction was used to amplify a 1.9-kb fragment, using FastStart Taq DNA Polymerase. PCR primers are located near the 5' end of the mRNA, so a PCR product (Figure 5) can be generated only when the full-length 14-kb mRNA is completely transcribed.
 


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 (See Figure 1 under "Typical Experiment" for details.)
  • 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.

 

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.

 

Product No: 03531317001
Product No: 03531295001
Product No: 03531287001