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Picture of Transcriptor First Strand cDNA Synthesis Kit

Transcriptor First Strand cDNA Synthesis Kit

For robust two-step RT-PCR and RT-qPCR of RNA up to 14 kb.

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

Product No. Pack Size List Price Your Price Quantity
04379012001
50 reactions $ 341.00

Product No. Pack Size List Price Your Price Quantity
04896866001
100 reactions $ 659.00

Product No. Pack Size List Price Your Price Quantity
04897030001
200 reactions $ 1,160.00

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 

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:

  • Studying 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.
  • Generating cDNA libraries with large and full-length inserts.
  • Cloning 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.



Figure 1: Quantification of the G6PDH gene with the Transcriptor First Strand cDNA Synthesis Kit and the LightCycler® 2.0 Instrument.

Different amounts of total RNA from K-562 cells were reverse transcribed using random hexamer primers and the reaction conditions outlined in the pack insert of the kit. The subsequent PCR was performed with primers specific for the G6PDH gene and the LightCycler® FastStart DNA Master SYBR Green I kit.
Blue curves: Transcriptor First Strand cDNA Synthesis Kit.
Red curves: RNase H mutant of the M-MuLV Reverse Transcriptase from Supplier A.
The Transcriptor First Strand cDNA Synthesis Kit produces curves with a better shape, higher fluorescence intensity, and lower crossing points, compared to the product from Supplier A. In addition, a straightforward analysis of the expression pattern of the G6PDH gene over a broader linear range and dynamic detection range is possible.




Figure 2: Quantification of the h-HPRT gene with the Transcriptor First Strand cDNA Synthesis Kit and the LightCycler® 480 Instrument.
Using the Transcriptor First Strand cDNA Synthesis Kit, total RNA was reverse transcribed by random hexamer priming; 100 ng/reaction to 10 pg/reaction of the transcribed RNA was used in a subsequent PCR. Using the LightCycler® 480 Instrument, eighteen-fold repeats of h-HPRT can be detected with high reproducibility (delta Cp ranging from 0.29 to 1.06, depending on the dilution).




Figure 3: Performance of the Transcriptor First Strand cDNA Synthesis Kit in two-step RT-PCR.
Total RNA was reverse transcribed using the anchored-oligo(dT)18 primer and reaction conditions outlined in the pack insert of the product. In the subsequent PCR, 5 µl of the cDNA reaction was used. After 30 cycles, the PCR products were separated on an agarose gel.
Lanes 1, 2: Starting material: Human skeletal muscle total RNA; Fragment amplified: Dystrophin gene; PCR Component: Expand High Fidelity PCR System.
Lane 3: Starting material: Human liver total RNA; Fragment amplified: Apo B gene; PCR Component: Expand High Fidelity PCR System.
Lane 4: Starting material: Human skeletal muscle total RNA; Fragment amplified: Dystrophin gene; PCR Component: Expand High Fidelity PCR System.
Lanes 5 - 9: Starting material: Human skeletal muscle total RNA; Fragment amplified: Dystrophin gene; PCR Component: Expand Long Template PCR System.



Figure 4: Altered expression of a broad range of protease genes in SH-SY5Y neuroblastoma cells after Bortezomib exposure.
RNA from two million neuroblastoma cells was isolated using the High Pure RNA Isolation Kit. cDNA was transcribed using the Transcriptor First Strand cDNA Synthesis Kit. qRT-PCR was performed on the LightCycler® 480 Instrument using the LightCycler® 480 Probes Master and RealTime ready Protease Custom Panel. Differences between Cp values of a Bortezomib-treated sample and the mean of six control assays were plotted. Cp values were normalized using five housekeeping genes. Bars represent SD (n=6).
Further experimental details are described in Cancer Research Application Note No. 2.
Data kindly provided by H. Barti-Juhász, Semmelweis University, Budapest, Hungary.

Reverse Transcriptase
Transcriptor Reverse Transcriptase – the core component of the kit – is a highly sensitive, highly thermostable, recombinant reverse transcriptase. The enzyme has RNA-directed DNA polymerase activity, DNA-dependent DNA polymerase activity, unwinding activity, and RNase H activity (for degrading RNA in RNA:DNA hybrids). The presence of the latter activity circumvents the need for an additional time-consuming RNase H incubation step after reverse transcription. Thus, Transcriptor Reverse Transcriptase is an ideal enzyme for first strand cDNA synthesis.The enzyme can be used at temperatures up to +65°C. Due to its thermostability, Transcriptor Reverse Transcriptase can accurately transcribe GC-rich templates that have large amounts of secondary structure. This property of Transcriptor Reverse Transcriptase eliminates the need for special reaction-enhancing additives.
Random hexamer primers
Random hexamer primers (included in the kit) bind throughout the entire length of RNA, ensuring reverse transcription of all RNA sequences. These primers also allow reverse transcription of RNAs that do not carry a poly(A) tail.
Anchored oligo(dT)18 primer
The anchored-oligo(dT)18 primer (included in the kit) is designed to bind at the beginning of the poly(A) tail (rather than randomly within the tail) to generate full-length cDNAs. Since the 5' ends of long mRNAs are often underrepresented in cDNA mixtures, this primer is preferred for most applications.
Protector RNase Inhibitor
Thermostable Protector RNase Inhibitor (included in the kit) protects RNA from degradation at high reaction temperatures.
 

Quality

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 µg 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 µl. The reaction is incubated for 1 hour at +50°C. In a subsequent PCR, 5 µl cDNA template is used in a total volume of 50 µl 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 µl; 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 µl 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.

Product No: 04379012001
Product No: 04896866001
Product No: 04897030001

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