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Using GC-rich templates for RT-PCR can lead to RNA template hybridization to newly synthesized cDNA, reducing sensitivity and specificity.
It is therefore important to use reverse transcriptase enzymes with an endogenous RNase H activity. Transcriptor Reverse Transcriptase has endogenous RNase H activity that efficiently digests RNA in RNA:DNA hybrids after cDNA synthesis, allowing PCR primers to more easily bind cDNA, which can increase PCR sensitivity.", "Country": "XG", "Code": "RNase H Activity", "Name": "RNase H Activity" }, { "Language": "en", "Value": "Up to 14 kb", "Country": "XG", "Code": "Maximum Fragment Size", "Name": "Maximum Fragment Size" }, { "Language": "en", "Value": "Store the kit at -15 to -25°C.", "Country": "XG", "Code": "Storage Conditions (Product)", "Name": "Storage Conditions (Product)" }, { "Language": "en", "Value": "+42 to +65°C", "Country": "XG", "Code": "Temperature Optimum", "Name": "Temperature Optimum" }, { "Language": "en", "Value": "No", "Country": "XG", "Code": "Proofreading Activity", "Name": "Proofreading Activity" }, { "Language": "en", "Value": "Kit", "Country": "XG", "Code": "Form of Supply", "Name": "Form of Supply" }, { "Language": "en", "Value": "Each lot of the kit is function tested using RT-PCR on a conventional thermal cycler, as well as the LightCycler® 2.0 Instrument. In addition, Transcriptor Reverse Transcriptase, Protector RNase Inhibitor, and the other kit components are tested independently for the absence of contamination, according to the current Quality Control procedures.

Function tested by 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 tested by two-step RT-PCR using the LightCycler® 2.0 Instrument and 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 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. Resulting curves must have specified crossing points and fluorescence intensities.", "Country": "XG", "Code": "Quality Control", "Name": "Quality Control" }, { "Language": "en", "Value": "SPECIAL PRECAUTIONS WHEN WORKING WITH RNA

  • Always wear gloves when working with RNA. After putting on gloves, do not touch surfaces and equipment to avoid reintroduction of RNases to decontaminated material.
  • Designate a special area for RNA work only.
  • Treat surfaces of benches and glassware with commercially available RNase inactivating agents. Clean benches with 100% ethanol.
  • Use commercially available sterile and RNase-free disposable plasticware only.
  • Purchase reagents that are free of RNases. Reserve separate reagents for RNA work only. Make all solutions using DEPC - treated water.
  • Keep all required reagents on ice.
  • Extract RNA as quickly as possible after obtaining samples. For best results, use either fresh samples or samples that have been quickly frozen in liquid nitrogen and stored at -60°C or below.
", "Country": "XG", "Code": "General Considerations", "Name": "General Considerations" }, { "Language": "en", "Value": "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 reverse transcribed in the presence of a primer.
Transcriptor Reverse Transcriptase is recommended for RT-PCR because of its high sensitivity 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. 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.", "Country": "XG", "Code": "Background Information", "Name": "Background Information" }, { "Language": "en", "Value": "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.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" }, { "Language": "en", "Value": "WORKING WITH DIFFICULT RNA TEMPLATES

When working with difficult RNA templates containing secondary structures, such as GC-rich templates, you can successfully reverse transcribe them using the following procedure:
  • Denature the template-primer mixture for 10 min at +65°C before adding the reverse transcriptase enzyme.
  • Use either random hexamer primers or gene-specific primers.
  • Use reverse transcriptases which allow reverse transcription at higher temperatures.

GENERATION OF FULL-LENGTH cDNA

For the generation of full length cDNA up to 14 kb, use anchored-oligo(dT) primers.

The fragment of interest is located at the 5' end of the mRNA

When the fragment of interest is located on the 5’ end, a mix of random hexamers and anchored oligo(dT)N can give optimal results. Typically, 60 μM random hexamers and 2.5 μM anchored oligo(dT)N are used. The Transcriptor First Strand cDNA Synthesis Kit includes both primer types, random hexamer primers and anchored oligo(dT) primers, as well as a detailed protocol for this type of application.

HANDLING OF RNA

  • Storage

Short-term storage:
Resuspended RNA is best stored at -20°C in RNase-free buffer TE buffer (10 mM Tris, 1mM EDTA) or RNase-free H2O (with 0.1 mM EDTA).
Note: It is important to use an EDTA solution known to be RNase-free for this purpose (older EDTA solutions may have microbial growth that can contaminate RNA sample with nucleases). RNA is generally stable at -80° C for up to a year without degradation.

Long term storage:
RNA samples can also be stored up to -80°C as ethanol precipitates.
Note: RNA is most stable in an NH4OAc/ethanol precipitation mixture* at -80°C (although, if necessary, RNA can also be resuspended in water or buffer for storage at -80°C).

Alternative storage of purified RNA:
Store RNA as a pellet in 70% ethanol or by adding 2 volumes of 100% ethanol to the resuspended RNA. Recover the RNA by adding sodium acetate and centrifuging.

Generally, it is recommended that RNA samples be aliquoted into several tubes. This will both prevent damage to the RNA due to successive freeze-thawing, and help to prevent inadvertent RNase contamination.

  • Recovering and Dissolving RNA

After centrifugation, RNA should be dried briefly at +37°C, or in a vacuum oven if available.
Note: Avoid completely drying the RNA pellet, because this makes RNA difficult to resuspend. When working with RNA, place all samples on ice. For reasons mentioned above, RNA is very susceptible to degradation at room temperature. Dissolve RNA by adding RNase-free buffer TE buffer (10 mM Tris, 1mM EDTA) or RNase-free H2O (with 0.1 mM EDTA); then incubate the tube on ice for 15 min. Gently tap or carefully vortex the tube to resuspend RNA.

  • Precipitation of RNA for downstream applications

When more concentrated purified RNA is required for downstream applications, an ammonium acetate (NH4OAc) precipitation (0.1 volumes of 5 M NH4OAc, and 2-2.5 volumes 100% ethanol, at -20°C for >25 min) results in excellent recovery of RNA. For quantitative recovery of low concentrations of RNA (ng/ml), an inert coprecipitant, such as glycogen, yeast RNA, or linear acrylamide, should be used.

  • Use of RNase Inhibitors

RNase inhibitors, such as Protector RNase Inhibitor are ideal to protect RNA from degradation both during isolation and purification, and also in downstream applications, such as reverse transcription and in vitro RNA transcription and translation.", "Country": "XG", "Code": "Protocols - Help Corner", "Name": "Protocols - Help Corner" }, { "Language": "en", "Value": "
Vial / BottleCapLabelFunctionCatalog NumberContent
1redTranscriptor  Reverse Transcriptase
 
Contains Transcriptor Reverse Transcriptase, 200 mM potassium phosphate, 2 mM  dithiothreitol, 0.2% Triton X-100 (v/v), 50% glycerol (v/v),  pH approximately 7.2043790120011 vial, 25 μl
04896866001 1 vial, 50 μl
048970300012 vials, each  50 μl
2colorlessTranscriptor  RT Reaction Buffer 5x conc.
 
Contains RT Reaction Buffer,  250 mM Tris/HCl, 150 mM KCl, 40 mM MgCl2, pH approximately 8.5 (+25°C)043790120011 vial. 1 ml
04896866001 1 vial, 1 ml
048970300012 vials, each 1 ml
3colorlessProtector RNase Inhibitor
 
Includes 20 mM Hepes-KOH, 50 mM KCl, 8mM dithiothreitol, 50% glycerol (v/v), pH approximately 7.6 (+4°C)043790120011 vial , 50 μl (40 U/μl)
048968660011 vial, 100 μl (40 U/μl)
048970300012 vials, each 100 μl (40 U/μl)
4yellow/
purple
purple
Deoxynucleotide Mix
 
10 mM each dATP, dCTP, dGTP, dTTP043790120011 vial, 100 μl
048968660011 vial, 200 μl
048970300012 vials, 200 μl
5blueAnchored-oligo(dT)18 Primer 043790120011 vial, 100 μl (50 μM)
048968660011 vial, 200 μl (50 μM)
048970300012 vials, each 200 μl  (50 μM)
6
 
blueRandom Hexamer Primer 043790120011 vial, 100 μl (600 μM)
048968660011 vial, 200 μl (600 μM)
048970300012 vials, each 200 μl  (600 μM)
For  Cat. No. 04896866001 and Cat. No. 04897030001
7colorlessWater, PCR GradeTo adjust the final volume
 In Cat. No. 04896866001 and Cat. No. 04897030001 the control reagents (Vial 7 and 8) are not included. In these kits Vial 7 is Water, PCR Grade.
048968660012 vials, each 1 ml
048970300013 vials, each 1 ml
For Cat. No. 04379012001
7greenControl RNAContains a stabilized solution of a total RNA fraction purified from an immortalized cell line (K562)043790120011 vial , 20 μl (50ng/μl)
8greenControl Primer Mix PBGDHuman porphobilinogen deaminase (PBGD)  forward and reverse primer to amplify a 151 bp fragment 043790120011 vial, 40 μl, (5 μM)
9colorlessWater, PCR GradeTo adjust the final volume
In Cat. No. 04896866 001 and Cat. No. 04897030001 the control reagents (Vial 7 and 8) are not included. In these kits Vial 7 is Water, PCR Grade.
043790120011 vial, 1ml
", "Country": "XG", "Code": "Content", "Name": "Content" }, { "Language": "en", "Value": "RNase H digestion

The degradation of the original RNA is crucial for cDNA quality in the subsequent PCR. Using GC-rich templates for RT-PCR can lead to RNA template hybridization to newly synthesized cDNA, reducing sensitivity and specificity.
It is therefore important to use reverse transcriptase enzymes with an endogenous RNase H activity. Transcriptor Reverse Transcriptase has endogenous RNase H activity that efficiently digests RNA in RNA:DNA hybrids. This circumvents the need to perform an additional time-consuming RNase H incubation step after reverse transcription, effectively shortening hands-on time and reducing costs.", "Country": "XG", "Code": "Background Information - Help Corner", "Name": "Background Information - Help Corner" }, { "Language": "en", "Value": "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.


 

", "Country": "XG", "Code": "Principle", "Name": "Principle" }, { "Language": "en", "Value": "PROBLEMS WITH GC-RICH TEMPLATES

GC-rich templates may form secondary structures that adversely affect the RT-PCR. Secondary structures may lead to a dissociation of the polymerase from the RNA template followed by an interruption of the reverse transcription reaction, producing shortened transcripts. A result can be that the required primer binding sites for the subsequent PCR reaction are missing. In some cases RNA may form loops, leading to non-transcribed RNA domains for which downstream analysis is not possible.

When working with difficult RNA templates with secondary structures, successful reverse transcription can be achieved as follows:
Perform an initial denaturation step for the primer / template mixture at +65°C (for 5 to 10 min), before adding the reverse transcriptase can often produce a more positive result. After the denaturation step, immediately cool the tubes on ice to avoid RNA renaturation.
Use higher RT incubation temperatures to help eliminate problems caused by RNA template secondary structures. Transcriptor Reverse Transcriptase can be used at temperatures up to +65°C.", "Country": "XG", "Code": "Troubleshooting - Help Corner", "Name": "Troubleshooting - Help Corner" }, { "Language": "en", "Value": "
Depending on the type of analysis, to which the cDNA is to be subjected, use one of three different priming methods described below: 

Anchored-oligo(dT)18 primer:
  • As anchored-oligo(dT)18 primers are specific to the small pool of poly(A)+ RNA in the whole total RNA pool (1 to 2%), the amount of cDNA resulting from reverse transcription reactions with anchored-oligo(dT)18 primers is considerably lower than with random hexamers. Anchored-oligo(dT)18 priming is recommended when performing RT-PCR for new mRNA targets. Anchored-oligo(dT)18 produces an RT-PCR product more consistently than random hexamers or gene-specific primers. 
  • Binds the very beginning of the poly(A) tail
  • Prevents priming from internal sites of the poly(A) tail
  • Generates full-length cDNA
  • Preferred priming method for most two-step RT-PCR
  • Available as part of the Transcriptor First Strand cDNA Synthesis Kit

Random hexamer primer:
  • In general, reverse transcribing 4 μg total RNA using random hexamers at a final primer concentration of 60 μM is sufficient. Increasing the concentration of hexamers for the transcription of 5 μg RNA may increase yield of small PCR products (< 500 bp), but may also decrease the yield of longer PCR products and full-length transcripts.
    Note that random hexamer priming is the mostly used non-specific priming method; specificity is only obtained using the types of PCR primers described below.
  • Binds many sites throughout the length of an RNA
  • Provides uniform representation of all RNA sequences in mRNA
  • Can prime cDNA transcription from RNAs that do not carry a poly(A)+ tail
  • The ratio of random primers to RNA in the RT reaction determines the average length ofcDNAs generated. Example: A high ratio will generate relatively short cDNAs, increasingthe chance of transcribing the entire target sequence.
    Short cDNA transcripts may help to overcome difficulties caused by RNA secondary structures.

Sequence-specific primer:
  • The use of gene-specific primers (recommended final concentration is 2 μM) is the most specific priming method, but this method can sometimes fail to prime cDNA even when the same primers are successful in PCR. If gene-specific priming fails in RT-PCR, repeat first-strand synthesis using anchored-oligo(dT)18 primers.
    Whenever possible, design primers that anneal to exon sequences on both sides of an intron or on exon/exon boundaries. This will allow differentiation of the amplified cDNA from contaminating genomic DNA because amplification of DNA will result in longer amplicons due to the additional intron sequence.
  • Primers shall not be self-complementary.
  • Binds only sequences that are exactly complementary to the primer sequence
  • Selects for a particular RNA
  • Greatly increases the specificity of the RT-PCR
", "Country": "XG", "Code": "Primers", "Name": "Primers" }, { "Language": "en", "Value": "
Standard laboratory equipment
  • Nuclease free, aerosol-resistant pipette tips
  • Pipettes with disposable, positive-displacement tips
  • Sterile reaction tubes for preparing PCR mixes and dilutions
  • Standard benchtop microcentrifuge
  • Standard block cycler instrument
For the RT-PCR reaction
  • sequence-specific PCR primers (optional)
  • Template RNA
  • PCR reaction vessels (thin-walled PCR tubes or plates are recommended)
  • For control reactions in combination with a LightCycler® 1.5 Instrument: LightCycler® 2.0 Instrument or LightCycler®480 Instrument
", "Country": "XG", "Code": "Additional Equipment and Reagent Required", "Name": "Additional Equipment and Reagent Required" }, { "Language": "en", "Value": "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.", "Country": "XG", "Code": "Applications", "Name": "Applications" }, { "Language": "en", "Value": "The control reaction which is provided in Cat.No. 04379012001 includes reverse transcription of the Control RNA followed by detection of a 151 bp fragment of PBGD in a PCR on a conventional thermal block cycler or a LightCycler® Instrument.", "Country": "XG", "Code": "Control Reactions", "Name": "Control Reactions" } ] } } ] }

Transcriptor First Strand cDNA Synthesis Kit

The Transcriptor First Strand cDNA Synthesis Kit is designed for first-strand cDNA synthesis reactions up to 14 kb.

Transcriptor First Strand cDNA Synthesis Kit

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