1 kit (50 rxns)
The Transcriptor First Strand cDNA Synthesis Kit is designed to reverse transcribe RNA (mRNA, total RNA, viral RNA, and in vitro -transcribed RNA) from a variety of sources for the following applications:
- Study gene expression levels, via two-step RT-PCR, using qualitative RT-PCR or quantitative RT-PCR on the LightCycler® Carousel-Based System, the LightCycler® 480 System, or other real-time instruments.
- Generate cDNA libraries with large and full-length inserts.
- Clone genes of interest.
The kit contains all components required for cDNA reactions for use with conventional thermal cyclers and real-time PCR instruments. In addition, the 50-reaction pack size includes 10 control reactions.
- 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.
- Transcribe a variety of templates, even the most difficult (e.g., GC-rich RNA with high secondary structures).
- Obtain cDNA transcripts up to 12 kb.
- Generate full-length transcripts with the anchored-oligo(dT)18 primer that is included in the kit.
- Achieve high reproducibility.
- Use three different priming methods – random hexamers, anchored-oligo(dT)18, and sequence-specific primers – depending on the type of analysis needed.
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 12 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.
- Transcriptor Reverse Transcriptase
- Transcriptor RT Reaction Buffer, 5x concentrated
- Protector RNase Inhibitor
- dNTP Mix, PCR-grade
- Anchored-oligo (dT)18 Primer
- Random Hexamer Primer
- Control RNA – total RNA fraction purified from the immortalized cell line K-562. Note: Included only in the 50-reaction pack size.
- Control PBGD Primer Mix (forward and reverse primer) Note: Included only in the 50-reaction pack size.
- Water, PCR-grade
Each lot of the kit is function tested in RT-PCR with a conventional thermal cycler as well as with the LightCycler® 2.0 Instrument. In addition, Reverse Transcriptase, Protector RNase Inhibitor, and the other kit components are tested independently for the absence of any contamination.
Function test in a two-step RT-PCR using a conventional thermal cycler: Transcriptor Reverse Transcriptase is function tested using 2 ìg of human skeletal muscle total RNA, 10 U Transcriptor Reverse Transcriptase, and 50 pmol anchored-oligo(dT)18 primer in a reaction volume of 20 ìl. The reaction is incubated for 1 hour at 50°C. In a subsequent PCR, 5 ìl cDNA template is used in a total volume of 50 ìl with the Expand Long Template PCR System to amplify a 10 kb dystrophin fragment. After 30 PCR cycles, the 10 kb fragment must be clearly visible after agarose-gel electrophoresis and ethidium bromide staining.
Function test in two-step RT-PCR using the LightCycler® 2.0 Instrument and the LightCycler® 480 Instrument: The kit is function tested using the supplied control. The control RNA (total RNA fraction from the immortalized K-562 cell line) is reverse transcribed with 10 U Transcriptor Reverse Transcriptase in a final reaction volume of 20 ìl; the reaction is incubated for 30 minutes at 55°C. Both hexamer primers and the anchored-oligo(dT)18 primer are tested. In subsequent quantitative PCRs, using the LightCycler® 2.0 Instrument and the LightCycler® 480 Instrument, 5 ìl of the cDNA reaction is incubated with the PBGD control primer mix and the LightCycler® FastStart DNA Master SYBR Green I or the LightCycler® 480 SYBR Green I Master. The resulting curves must have defined crossing points and fluorescence intensities.