Transcriptor Reverse Transcriptase – the core component of the kit – is a recombinant reverse transcriptase expressed in?E. coli. The enzyme has RNA-directed DNA polymerase activity, DNA-dependent DNA polymerase activity, unwinding activity, and RNase H activity that degrades RNA in RNA:DNA hybrids. The latter circumvents the need to perform an additional time-consuming RNase H incubation step after reverse transcription. This shortens the reaction time and reduces costs.
Transcriptor Reverse Transcriptase is recommended for RT-PCR because of its high sensitivity in combination with high thermostability: the enzyme synthesizes long cDNA products (up to 14 kb) and can be used at temperatures up to +65°C. Due to its thermostability, Transcriptor Reverse Transcriptase is recommended for GC-rich templates with high secondary structure, without the need to include additives in the reaction.
The kit provides all reagents required for first-strand cDNA synthesis reactions. For priming, three different primer systems can be used. Two cDNA synthesis primers are provided with the kit: random hexamer primers and an anchored-oligo(dT)18?primer. The latter is designed to bind at the beginning of the poly(A) tail to generate full-length cDNAs and to prevent priming from internal sites of the poly(A) tail. The 5' ends of long mRNAs are often underrepresented; therefore, this priming method is preferred for most applications. The use of random hexamer primers enables priming throughout the length of RNA for uniform representation of all RNA sequences and allows reverse transcription of RNAs that do not carry a poly(A) tail.
Thermostable Protector RNase Inhibitor is included in the kit to protect RNA from degradation at high reaction temperatures.

Optimized for Many Applications

• Use with conventional thermal cyclers and real-time PCR instruments.
• Optimize real-time PCR – the kit is tested with the LightCycler??Carousel-Based System, the LightCycler??480 System, and other real-time instruments.
• Obtain accurate linear quantification over at least a 108?-fold range of input RNA (in vitro?transcripts), permitting the analysis of genes with very low or extremely high expression levels.
• Generate efficient qPCR curves, with high fluorescence intensity and identical distances between RNA dilutions, allowing a straightforward analysis of results (Figure 1).
• Obtain reliable and sensitive results – there are no additives in the RT buffer system that interfere with – or inhibit – the subsequent PCR.

Robustness

• Transcribe a variety of templates, even the most difficult (e.g.,?GC-rich RNA with high secondary structures).
• ?Achieve high reproducibility (Figure 2).
• Generate full-length transcripts with the anchored-oligo(dT)18?primer that is included in the kit.
• Obtain cDNA transcripts up to 14 kb (Figure 3).

Flexibility

• Use three different priming methods – random hexamers, anchored-oligo(dT)18, and sequence-specific primers – depending on the type of analysis needed.

Contents

1. Transcriptor Reverse Transcriptase
2. Transcriptor RT Reaction Buffer, 5x concentrated
3. Protector RNase Inhibitor
4. dNTP Mix, PCR Grade
5. Anchored Oligo (dT)18?Primer
6. Random Hexamer Primer?
7. Control RNA – total RNA fraction purified from the immortalized cell line K-562.
   Note:?Included only in the?50-reaction pack size
8. Control PBGD Primer Mix (forward and reverse primer)
   Note: Included only in the?50-reaction pack size
9. Water, PCR Grade?