Transcriptor First Strand cDNA Synthesis Kit
The Transcriptor First Strand cDNA Synthesis Kit provides all reagents required for first-strand cDNA synthesis reactions up to 14kb on all real-time instruments and conventional thermal cyclers. Three different priming methods can be used. The enclosed anchored-oligo(dT)18 primer is designed to bind at the beginning of the poly(A) tail and guarantees full-length cDNA synthesis. Transcriptor Reverse Transcriptase – the core component of the kit – has RNA-directed DNA polymerase activity, DNA-dependent DNA polymerase activity, unwinding activity, and RNase H activity that digests 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. Thermostable Protector RNase Inhibitor is included in the kit to protect RNA from degradation at high reaction temperatures.
Benefits of the Transcriptor First Strand cDNA Synthesis Kit
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.
- 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.
- Generate full-length transcripts with the anchored-oligo(dT)18 primer that is included in the kit.
- Obtain cDNA transcripts up to 14 kb.
Flexibility
Use three different priming methods – random hexamers, anchored-oligo(dT)18, and sequence-specific primers – depending on the type of analysis needed.
How the Transciptor First Strand cDNA Synthesis Kit works
Using the Transcriptor First Strand cDNA Synthesis Kit, RNA is reverse transcribed into single-stranded cDNA, which can be used directly for subsequent PCR with gene-specific primers on conventional thermal block cyclers and real-time PCR instruments (e.g. the LightCycler® Carousel-Based System, the LightCycler® 480 Instrument or other real-time PCR instruments), or for other downstream applications.
Transcriptor Reverse Transcriptase 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, very importantly, RNase H activity that degrades RNA in RNA:DNA hybrids. Thus, there is no need to perform an additional time-consuming RNase H incubation step after reverse transcription, significantly shortening the reaction time. Single-stranded RNA as well as ssDNA are accepted as template and are reverse transcribed in the presence of a primer.
Transcriptor Reverse Transcriptase is recommended for RT-PCR because of its high sensi-tivity in connection with very high thermostability: The enzyme is able to synthesize long cDNA products (up to 14 kb) and can be used at temperatures up to +65°C. Due to its high 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 performing first strand cDNA synthesis reactions from RNA. For priming, three different primer systems may be used as shown in Figure 5. Two cDNA synthesis primers are already provided with the kit: random hexamer primers and an anchored-oligo(dT)18 primer. The latter is designed to bind at the very beginning of the poly(A) tail to generate full-length cDNA and to prevent priming from internal sites of the poly(A) tail. Although 5′-ends of long mRNAs can be underrepresented, this priming method is preferred for most applications.. The use of random hexamer primers provides priming throughout the length of RNA for uniform representation of all RNA sequences and, in addition, allows reverse transcription of RNA molecules that do not have a poly(A) tail.
Figure: Control reaction with PBGD primers using LightCylcer ® FastStart DNA Master SYBR Green I for the LightCycler ® Carousel-Based Instrument reaction. The supplied Control RNA was used in different diluti
