( ...)Increase the Power and Sensitivity for Your cDNA,,,Synthesis with the New Transcriptor Reverse Transcriptase

Transcriptor Reverse Transcriptase is a new recombinant reverse transcriptase (RT) expressed in E. coli. The enzyme has RNA-directed DNA polymerase activity, DNA-dependent DNA polymerase activity, unwinding activity, and, importantly, RNase H activity.

Due to the special recombinant structure of Transcriptor Reverse Transcriptase, the individual enzymatic activities are resulting in a number of unique and beneficial properties.

Achieve High Sensitivity

• In two-step RT-PCR with the LightCycler Instrument,
  

Transcriptor Reverse Transcriptase has a broad dynamic detection range of 12 1.2 x 109 copies of in vitro transcribed RNA (Figure 1). This enables analysis of genes with very low or extremely high expression levels.

A linear quantification over 108-fold range of input RNA can be performed with the same efficiency with Transcriptor Reverse Transcriptase, making expression analysis of induced or repressed genes possible (Figure 1).

The high sensitivity of Transcriptor Reverse Transcriptase compared to other reverse transcriptases improves the performance of existing assays and allows performance of assays that were not previously possible (Figure 2).

High fluorescence intensities and well-shaped curves with identical distances for cDNA dilutions lead to clear and interpretable results compared to competitor products (Figure 2).

• In two-step RT-PCR using conventional thermal cyclers,
  

As little as 100 pg of total RNA is detected, in combination with the new FastStart High Fidelity PCR System. This enables the analysis of low expressed RNA and RNA when sample material is limited (Figure 3).

• In one-step RT-PCR using conventional thermal cyclers,

As little as 1 pg of total RNA or 100 fg mRNA is detected, in combination with the Expand High Fidelity PCR System. This permits the analysis of low expressed RNA in one-step RT-PCR.

Obtain Full-Length cDNA Synthesis

cDNA up to 14 kb in length can be generated with Transcriptor Reverse Transcriptase, making it the product of choice for reverse transcription of very long eukaryotic mRNA, as shown in Figure 4 for human dystrophin, one of the longest mRNAs with a length of 14 kb. To achieve high sensitivity and yield in the following PCR, use FastStart Taq DNA Polymerase or the new FastStart High Fidelity Enzyme Blend. Both allow hot-start PCR and are available as single reagents or included in different LightCycler Kits (only Fast Start Taq DNA Polymerase). For amplification of longer fragments, the Expand Long Template PCR System is recommended.

Obtain the Power to Reverse Transcribe the Most Difficult RNAs

Due to the very high thermostability of Transcriptor Reverse Transcriptase (up to 65C) compared to other thermostable reverse transcriptases, robust reverse transcription of GC-rich RNA with high secondary structure can now be easily achieved (Figure 5). In combination with FastStart Taq DNA Polymerase and the GC-RICH Resolution Solution, Transcriptor Reverse Transcriptase is the product of choice when difficult RNAs must be reverse transcribed and amplified in a subsequent PCR.

Rely on a Multi-Purpose Enzyme Tested in a Wide Variety of Assays

Transcriptor Reverse Transcriptase shows premium performance with conventional thermal cyclers and the LightCycler Instrument. Compared to other reverse transcriptases, it is produced under strict regulatory conditions and function-tested in both systems ensuring functionality in end-point and real-time PCR. Additional specifications, such as bioburden, increase the safety level when working with RNA.

Efficiently Label RNA

Efficient incorporation of modified nucleotides during cDNA synthesis is possible, making Transcriptor Reverse Transcriptase an important labeling enzyme in, for example, microarray applications. Labeling with Cyanine 3-, Cyanine 5-, aminoallyl-, DIG-, and Biotin-dUTP was tested; the result was high-efficiency incorporation of these modified nucleotides.

Shorten the Reaction Time

With Transcriptor Reverse Transcriptase there is no need to perform an extra RNase H incubation step following cDNA synthesis as recommended to remove RNA complementary to cDNA when using a RNase H-- mutated reverse transcriptase. RNase H--mutated reverse transcriptase can limit the sensitivity of RT-PCR detection as shown for the gene NCX2, which was detected much more readily when a RNase H step was included after the reverse transcription [1]. If the RNA template is not degraded after first-strand cDNA synthesis, it can bind to the newly synthesized cDNA and restrict its accessibility to primers during subsequent PCR amplification. RNase H-mediated destruction of the template can prevent this problem and improve the sensitivity of RT-PCR analysis.

Additionally, Transcriptor is a fast reverse transcriptase, requiring only 30 minutes for first-strand cDNA synthesis, which results in a reduction of the total reaction time.

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

• Synthesis of first-strand cDNA for use in subsequent amplification reactions

• RT-PCR of GC-rich RNA templates

• Incorporation of Cyanine 3-, Cyanine 5-, DIG-, Biotin-, and aminoallyl-modified nucleotides during cDNA synthesis

• Retrieval and cloning of the 5′ and 3′ termini of mRNA by RACE (Rapid Amplification of cDNA Ends)

• Generation of cDNA libraries with large inserts.

Transcriptor Reverse Transcriptase is supplied in storage buffer with a volume activity of 20 U/l. A 5x concentrated reaction buffer is also provided.

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