UltraScript® Reverse Transcriptase & cDNA Synthesis Kits

UltraScript® Reverse Transcriptase is a robust and thermostable modified MMLV reverse transcriptase engineered to enhance cDNA synthesis speed and yield with accurate transcript representation.

The enzyme allows highly sensitive reverse transcription of viral RNA sequences, making it an ideal component of RT-qPCR-based detection tests. Available as a stand-alone enzyme, or as a ready mix comprising reverse transcriptase, buffer system and, optionally, with an optimised blend of random hexamers with anchored oligo(dT) primers. This reverse transcriptase provides unbiased, efficient and sensitive cDNA synthesis over a broad range of RNA template concentrations. 

Features

  • Unbiased representation of 5’ and 3’ mRNA transcript ends
  • Reduced RNase H activity 
  • Thermostable reverse transcriptase 45 °C to 55 °C
  • Advanced RNase inhibitor
  • High cDNA yields from as little as 4 pg total RNA
  • Accurate reverse transcription of GC-rich templates
  • Sensitive detection of low copy number transcripts
  • Advanced buffer chemistry including Mg and dNTPs
  • Both premixed or separate oligo cDNA synthesis kits available
CAT No.
Product
Size
Price
QTY
PB30.11-02
UltraScript® cDNA Synthesis Kit
25 x 20 μL Reactions
$151.00
PB30.11-10
UltraScript® cDNA Synthesis Kit
100 x 20 μL Reactions
$516.00
PB30.12-01
UltraScript® Reverse Transcriptase
10 000 Units
$238.00
PB30.12-04
UltraScript® Reverse Transcriptase
40 000 Units
$849.00
PB30.13-01
UltraScript® Reverse Transcriptase (8000 U/μL)
80 000 Units
PB30.15-02
UltraScript® cDNA Synthesis Kit Separate Oligos
25 x 20 μL Reactions
$151.00
PB30.15-10
UltraScript® cDNA Synthesis Kit Separate Oligos
100 x 20 μL Reactions
$516.00

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More Information

UltraScript® Reverse Transcriptase is a thermostable and extremely active modified MMLV reverse transcriptase (RTase) for cDNA synthesis1. The reaction temperature can be increased up to 55 °C providing higher specificity and efficient transcription of GC-rich RNA regions with a high secondary structure. The RTase is blended with an advanced RNase inhibitor preventing degradation of RNA by contaminating RNase. UltraScript® Reverse Transcriptase is not inhibited by ribosomal and transfer RNAs, making total RNA an ideal substrate.

The enzyme is supplied with a 5x buffer containing Mg, dNTPs, stabilizers and enhancers, optimised to generate high yield cDNA from as little as 4 pg RNA. As oligos are not included, UltraScript® Reverse Transcriptase provides the convenience and flexibility for users to define their own priming strategy depending on the type of analysis needed. UltraScript® Reverse Transcriptase gives exceptional performance with gene-specific primers, oligo(dT) and random hexamers to produce high quality cDNA ideal for a variety of downstream applications.

Our powerful RTase is also available in high concentration formats, and as ready-made UltraScript® cDNA Synthesis Kit (formerly qPCRBIO cDNA Synthesis Kit), in which the relative concentrations of random hexamers and anchored oligo(dT) have been optimised for the generation of cDNA for use in real-time PCR experiments. This eliminates the need for user optimisation of the critical reverse transcription reaction factors, such as priming strategy, reverse transcriptase, buffer and enhancers, and gives unbiased, efficient and sensitive cDNA synthesis every time.

Read these Tips and Tricks to make the most of your cDNA synthesis experiments. For a quick summary of when each cDNA synthesis product is available use the reference table below:

Primers

RNA Source

Intended Use

Recommended Product

Oligo(dT) & Random hexamers Any qPCR, cloning, NGS PB30.11/PB30.15
Random hexamers Prokaryotic, Archaeal qPCR, cloning, NGS PB30.15, (PB30.12 user must supply primer)
Oligo(dT) Eukaryotic qPCR, cloning, NGS PB30.15, (PB30.12 user must supply primer)
Gene specific primer Any Cloning, targeted qPCR,        targeted NGS PB30.12 (user must supply primer)

Applications

  • Random hexamer, oligo(dT) and gene-specific primers
  • cDNA synthesis for real-time PCR analysis
  • cDNA synthesis for PCR analysis, cloning, library preparation and Next Generation Sequencing
  • Low copy number transcripts
  • Viral RNA targets
  • miRNA targets
  • Efficient synthesis from total RNA or poly(A)+ RNA

Specifications

UltraScript® cDNA Synthesis Kit

Component

25 Reactions

100 Reactions

20x UltraScript for cDNA Synthesis

1 x 25 μL

1 x 100 μL

5x cDNA Synthesis Mix

1 x 100 μL

4 x 100 μL

UltraScript® cDNA Synthesis Kit Separate Oligos

Component

25 Reactions

100 Reactions

20x UltraScript for cDNA Synthesis

1 x 25 μL

1 x 100 μL

5x UltraScript Buffer

1 x 200 μL

2 x 200 μL

100 μΜ Anchored Oligo(dT)18

1 x 100 μL

1 x 100 μL

100 μM Random Hexamers

1 x 100 μL

1 x 100 μL

UltraScript® Reverse Transcriptase

Component

10,000 Units

40,000 Units

UltraScript (200u/μL) with RNase inhibitor

2 x 25μL

2 x 100μL

5x UltraScript Buffer

1 x 200μL

4 x 200μL

UltraScript® Reverse Transcriptase (2500 U/μL)

Component

50 000 Units

UltraScript Reverse Transcriptase (2500 U/μL) with RNase Inhibitor

1 x 20 μL

Glycerol-Free Enzyme Dilution Buffer

1 x 1.5 mL

UltraScript® Reverse Transcriptase (8000 U/μL)

Component

80 000 Units

UltraScript Reverse Transcriptase (8000 U/μL)

1 x 10 μL

Glycerol-Free Enzyme Dilution Buffer

1 x 1.5mL

UltraScript® cDNA Synthesis Kit

Component

20x UltraScript for cDNA Synthesis

5x cDNA Synthesis Mix



25 Reactions

1 x 25 μL

1 x 100 μL

100 Reactions

1 x 100 μL

4 x 100 μL

UltraScript® cDNA Synthesis Kit Separate Oligos

Component

20x UltraScript for cDNA Synthesis

5x UltraScript Buffer

100 μΜ Anchored Oligo(dT)18

100 μM Random Hexamers



25 Reactions

1 x 25 μL

1 x 200 μL

1 x 100 μL

1 x 100 μL

100 Reactions

1 x 100 μL

2 x 200 μL

1 x 100 μL

1 x 100 μL

UltraScript® Reverse Transcriptase

Component

UltraScript (200u/μL) with RNase inhibitor

5x UltraScript Buffer



10,000 Units

2 x 25μL

1 x 200μL

40,000 Units

2 x 100μL

4 x 200μL

UltraScript® Reverse Transcriptase (2500 U/μL)

Component

UltraScript Reverse Transcriptase (2500 U/μL) with RNase Inhibitor

Glycerol-Free Enzyme Dilution Buffer



50 000 Units

1 x 20 μL

1 x 1.5 mL

UltraScript® Reverse Transcriptase (8000 U/μL)

Component

UltraScript Reverse Transcriptase (8000 U/μL)

Glycerol-Free Enzyme Dilution Buffer



80 000 Units

1 x 10 μL

1 x 1.5mL

Reaction Volume

Storage

Not applicable

On arrival, products should be stored between -30 and -15°C. If stored correctly the kit will retain full activity for 12 months.

Reaction Volume

Not applicable

Storage

On arrival, products should be stored between -30 and -15°C. If stored correctly the kit will retain full activity for 12 months.

FAQs

Can long targets (15-20 kb) be reverse transcribed by UltraScript™ RTase?

20 kb RNA reverse transcription can be achieved with all our RTases because RTases jump on and off their primed substrates. Secondary structures, unspecific binding and mis-priming can be the most common causes for the reaction ending prematurely.

We advise using specific primers or oligo(dT) and optimising each aspect of the reaction, for example, the different enzyme concentrations, primers, primer concentration, temperature and temperature cycling and time. Also, the subsequent PCR/qPCR step might need optimisation.

We recommend UltraScript™ 2.0 RTase for reverse transcribing long templates because it can sustain higher temperatures for longer, which may be necessary as the probability of secondary structures in RNA increases with its length.

We’ve included the following paper for your reference1.

1  Thiel, V. et al. Effective amplification of 20-kb DNA by reverse transcription PCR. Anal Biochem 252, 62-70, doi:10.1006/abio.1997.2307 (1997).

Can miRNA be amplified using UltraScript™ cDNA Synthesis Kit?

miRNA should not be amplified using UltraScript™ cDNA Synthesis Kit. UltraScript™ cDNA Synthesis Kit Separate Oligos, UltraScript Reverse Transcriptase, or UltraScript 2.0 Reverse Transcriptase should be used because they come with a buffer that does not contain random hexamers and oligod(T)s, which could interfere with miRNA specific primers.

Can miRNA be amplified using UltraScript™ Reverse Transcriptase?

All our RTases are can be used for miRNA quantification and analysis. However, we do not sell any dedicated kits.

We advise that you use one of the two following approaches:

  • Use universal RT primers and add poly(A) or poly(U) tails (e.g. by poly(U)-polymerase), followed by cDNA synthesis using universal primers1,2.
  • Use specific RT primers and omit the tailing step1,3-5.

If you are unfamiliar with the specifics of those approaches, please refer to the reference list below, which serve as a guideline.

1  Dave, V. P. et al. MicroRNA amplification and detection technologies: opportunities and challenges for point of care diagnostics. Lab Invest 99, 452-469, doi:10.1038/s41374-018-0143-3 (2019).

2  Mei, Q. et al. A facile and specific assay for quantifying microRNA by an optimized RT-qPCR approach. PLoS One 7, e46890, doi:10.1371/journal.pone.0046890 (2012).

3  Chen, C. et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33, e179, doi:10.1093/nar/gni178 (2005).

4  Raymond, C. K., Roberts, B. S., Garrett-Engele, P., Lim, L. P. & Johnson, J. M. Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. RNA 11, 1737-1744, doi:10.1261/rna.2148705 (2005).

5  Androvic, P., Valihrach, L., Elling, J., Sjoback, R. & Kubista, M. Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification. Nucleic Acids Res 45, e144, doi:10.1093/nar/gkx588 (2017).

Can UltraScript™ cDNA Synthesis Kit be used for endpoint and qPCR?

All our RTases and kits can be used for both endpoint and qPCR applications.

Can UltraScript™ cDNA Synthesis Kit reverse transcribe eukaryotic and prokaryotic RNA?

UltraScript™ DNA Synthesis Kit can amplify both eukaryotic and prokaryotic RNA. Our mix contains random hexamers that are universal primers for all types of RNA, provided it contains sufficiently long fragments. Due to the presence of hexamers, RNA from all organisms can be reverse transcribed if it is available for the RTase. There are also oligo-d(T) included in the mix, which work with A-tailed RNA such as eukaryotic mRNA.

For a more selective kit use UltraScript™ Reverse Transcriptase or UltraScript™ 2.0 Reverse Transcriptase as the buffer for these products come without any hexamers and oligo(dT)s. This allows you to determine your own priming strategy.

Can UltraScript™ Reverse Transcriptase be used with blood or plasma samples?

Yes, however we recommend comparing it against UltraScript™ 2.0 for your applications since it is better-suited to handle the various inhibitors present in blood. We also recommend optimisation by performing a titration of the amount of blood/plasma sample for RT reaction.

Can UltraScript™ Reverse Transcriptase do template switching?

All our RTases can be used in template switching applications. We currently do not provide a full protocol or dedicated kit but we recommend referring to this list of articles for reference1-4.

1  Takada, S. & Mano, H. Profiling of microRNA expression by mRAP. Nat Protoc 2, 3136-3145, doi:10.1038/nprot.2007.457 (2007).

2  Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171-181, doi:10.1038/nprot.2014.006 (2014).

3  Moldovan, N. et al. Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus. Front Microbiol 8, 2708, doi:10.3389/fmicb.2017.02708 (2017).

4  Zajac, P., Islam, S., Hochgerner, H., Lonnerberg, P. & Linnarsson, S. Base preferences in non-templated nucleotide incorporation by MMLV-derived reverse transcriptases. PLoS One 8, e85270, doi:10.1371/journal.pone.0085270 (2013).

Is a thermocycler required to perform reverse transcription?

Reverse transcription is an isothermal process so any instrument that can maintain stable temperature will be fine. We do not advise using water baths due to contamination. A thermocycler has the added advantage of a heated lid that circumvents the problem of tube cap condensation.

What are the main differences between UltraScript™ and UltraScript™ 2.0 Reverse Transcriptases?

Both products are derived from wild-type MMLV reverse transcriptase and contain multiple mutations that enhance their function. They are each excellent RTases in their own right. UltraScript™ 2.0 is more thermostable up to 65 °C and above, resistant to inhibitors and capable of reverse transcribing large quantities of RNA up to 3.5 μg. UltraScript™ 2.0 can be also used for applications requiring sensitivity due to a low amount of RNA present, however, this requires substantial optimisation on the users side using the current formulation and we recommend using UltraScript™ instead. UltraScript™ can be used with 1-step kits whereas UltraScript™ 2.0 should be used only as a part of the 2-step process (RT then PCR). UltraScript™ 2.0 is especially recommended for long and difficult templates that contain large amounts secondary structure. Both RTases can be used with cDNA synthesis kit with/or without hexamers and oligo-d(T).

What is the dynamic range of UltraScript™ Reverse Transcriptase?

UltraScript™ RTase can transcribe as little as 4 pg and up to 0.4 μg of RNA. The performance depends on the type of RNA, RNA quality and whether specific primers, oligod(Ts) or random hexamers are used. For higher capacity applications please use UltraScript™ 2.0 Reverse Transcriptase and cDNA Synthesis Kits.

What is the error rate of UltraScript™ Reverse Transcriptase?

Like the great majority of the RTases available on the market UltraScript™ Reverse Transcriptase, as well as all other RTases we offer, are derived from wild-type Moloney Murine Leukemia Virus (MMLV) RTase and have an error rate of 1×10-4 errors/bp1. This also applies to RTases derived from Avian Myeloblastosis Virus (AMV).

Running the reaction at a higher temperature increases fidelity because it destabilises mismatched base pairs2. If low fidelity is required adding manganese will make RTases highly mutagenic and could increase their speed3.

1  Yasukawa, K. et al. Next-generation sequencing-based analysis of reverse transcriptase fidelity. Biochem Biophys Res Commun 492, 147-153, doi:10.1016/j.bbrc.2017.07.169 (2017).

2  Malboeuf, C. M., Isaacs, S. J., Tran, N. H. & Kim, B. Thermal effects on reverse transcription: improvement of accuracy and processivity in cDNA synthesis. Biotechniques 30, 1074-1078, 1080, 1082, passim, doi:10.2144/01305rr06 (2001).

3  Cadwell, R. C. & Joyce, G. F. Randomization of genes by PCR mutagenesis. PCR Methods Appl 2, 28-33 (1992).

What is the maximum length of product possible using UltraScript™ cDNA Synthesis Kit?

The RTase included in UltraScript™ cDNA Synthesis Kit can transcribe very long targets of up to 20 kB with sufficient optimisation1. However, the kit contains random hexamers that will align in random places along the sequence and this will greatly reduce the maximum length of a continuous product. Most of the products will be discontinuous to a large extent nonetheless, the whole sequence of RNA will be covered and subsequently be quantifiable with qPCR. This applies to any cDNA synthesis kit that incorporates random hexamers. If full-length continuous products are required, we recommend UltraScript™ Reverse Transcriptase or UltraScript™ 2.0 Reverse Transcriptase as these products come with a buffer without any hexamers and oligo(dT)s.

1  Thiel, V. et al. Effective amplification of 20 kb DNA by reverse transcription PCR. Anal Biochem 252, 62-70, doi:10.1006/abio.1997.2307 (1997).

What troubleshooting is there for low cDNA yield?

There could be multiple reasons for low yield occurring:

  • The amount or the integrity of your RNA could be insufficient to reach a significant amount of product required for the subsequent endpoint or qPCR step. Try increasing the amount RNA and/or check the quality of your RNA using gel electrophoresis and RIN value. Consider an alternative purification protocol for your RNA and/or use RNase inhibitor early on. All our RTases contain RNase inhibitor but for some applications it might be necessary to add it before RT step
  • You RNA prep may contain inhibitors such as haem, high concentrations of NaCl, SDS, guanidine thiocyanate, melanin, or calcium1. Try an alternative method of purification that gets rid of all substances that can inhibit reaction. You could also try diluting your RNA before the reaction. This will decrease the theoretical yield, but it will also dilute inhibitors to levels more permissible for the RTase to work efficiently. This could still provide enough product for subsequent steps.
  • Some RNAs are inherently difficult to transcribe due to secondary structures and/or stretches of sequences that are not an ideal substrate for RTase. Try increasing the temperature and/or performing RNA denaturation/primer annealing, at 70°C for 10 minutes, before adding RTase.
  • Using RNAseH treatment after RT step can increase the yield, especially for GC rich targets because RNA-DNA heteroduplexes are more stable than DNA-DNA duplexes.
  • If your sequences are still underrepresented it might be necessary to use a more targeted approach with specific primers or just oligo(dT)s. We recommend UltraScript Reverse Transcriptase or UltraScript 2.0 Reverse Transcriptase that come with a buffer without any hexamers and oligo(dT)s for that purpose.

1  Schrader, C., Schielke, A., Ellerbroek, L. & Johne, R. PCR inhibitors – occurrence, properties and removal. J Appl Microbiol 113, 1014-1026, doi:10.1111/j.1365-2672.2012.05384.x (2012).

What troubleshooting is there if smears or non-specific products are visible after using UltraScript™ RTase?

There could be multiple reasons for the appearance of smears or non-specific products after the RT reaction. The following points can be considered for troubleshooting:

  • It’s important to establish if the problem relates to the reverse transcription step or PCR/qPCR step, provided the PCR/qPCR step is performed downstream from the reverse transcription step. Use proper controls and troubleshoot the PCR/qPCR reaction to exclude this from the list of potential steps causing the above problems.
  • Smearing can indicate primer oligomerisation. Oligomerisation can be caused if the oligomers are badly designed and if the template concentration is low. This will result in reverse transcription of primer-dimers or off-target sequences. We advise redesigning primers, increasing reaction temperature, increasing the amount of template, decreasing primer concentration or shortening the reaction time. These considerations can also be used for non-specific bands.
  • Primers may not be specific to your target sequence and may require redesign.
  • Primers against highly repetitive sequences can cause smearing and redesigning your primers may have to be considered.
  • Your RNA may be degraded. Try increasing the amount of RNA and/or check the quality of your RNA using gel electrophoresis and RIN value. Try an alternative purification protocol for your RNA and/or use RNase inhibitor early on. All our RTases contain RNA inhibitor but for some applications it might be necessary to add it before the RT step.
  • Consider the standard precautions when handling RNA such as using gloves, positive displacement pipettes with aerosol barrier, etc.
  • Ensure there is no DNA contamination.

Why is there is a false positive in the RT-qPCR analysis?

Your RNA sample might be contaminated with DNA. We suggest re-purifying your RNA using a better protocol or use thermolabile DNase to get rid of any contamination.

More Information

UltraScript® Reverse Transcriptase is a thermostable and extremely active modified MMLV reverse transcriptase (RTase) for cDNA synthesis1. The reaction temperature can be increased up to 55 °C providing higher specificity and efficient transcription of GC-rich RNA regions with a high secondary structure. The RTase is blended with an advanced RNase inhibitor preventing degradation of RNA by contaminating RNase. UltraScript® Reverse Transcriptase is not inhibited by ribosomal and transfer RNAs, making total RNA an ideal substrate.

The enzyme is supplied with a 5x buffer containing Mg, dNTPs, stabilizers and enhancers, optimised to generate high yield cDNA from as little as 4 pg RNA. As oligos are not included, UltraScript® Reverse Transcriptase provides the convenience and flexibility for users to define their own priming strategy depending on the type of analysis needed. UltraScript® Reverse Transcriptase gives exceptional performance with gene-specific primers, oligo(dT) and random hexamers to produce high quality cDNA ideal for a variety of downstream applications.

Our powerful RTase is also available in high concentration formats, and as ready-made UltraScript® cDNA Synthesis Kit (formerly qPCRBIO cDNA Synthesis Kit), in which the relative concentrations of random hexamers and anchored oligo(dT) have been optimised for the generation of cDNA for use in real-time PCR experiments. This eliminates the need for user optimisation of the critical reverse transcription reaction factors, such as priming strategy, reverse transcriptase, buffer and enhancers, and gives unbiased, efficient and sensitive cDNA synthesis every time.

Read these Tips and Tricks to make the most of your cDNA synthesis experiments. For a quick summary of when each cDNA synthesis product is available use the reference table below:

Primers

RNA Source

Intended Use

Recommended Product

Oligo(dT) & Random hexamers Any qPCR, cloning, NGS PB30.11/PB30.15
Random hexamers Prokaryotic, Archaeal qPCR, cloning, NGS PB30.15, (PB30.12 user must supply primer)
Oligo(dT) Eukaryotic qPCR, cloning, NGS PB30.15, (PB30.12 user must supply primer)
Gene specific primer Any Cloning, targeted qPCR,        targeted NGS PB30.12 (user must supply primer)

Applications

  • Random hexamer, oligo(dT) and gene-specific primers
  • cDNA synthesis for real-time PCR analysis
  • cDNA synthesis for PCR analysis, cloning, library preparation and Next Generation Sequencing
  • Low copy number transcripts
  • Viral RNA targets
  • miRNA targets
  • Efficient synthesis from total RNA or poly(A)+ RNA

Specifications

UltraScript® cDNA Synthesis Kit

Component

25 Reactions

100 Reactions

20x UltraScript for cDNA Synthesis

1 x 25 μL

1 x 100 μL

5x cDNA Synthesis Mix

1 x 100 μL

4 x 100 μL

UltraScript® cDNA Synthesis Kit Separate Oligos

Component

25 Reactions

100 Reactions

20x UltraScript for cDNA Synthesis

1 x 25 μL

1 x 100 μL

5x UltraScript Buffer

1 x 200 μL

2 x 200 μL

100 μΜ Anchored Oligo(dT)18

1 x 100 μL

1 x 100 μL

100 μM Random Hexamers

1 x 100 μL

1 x 100 μL

UltraScript® Reverse Transcriptase

Component

10,000 Units

40,000 Units

UltraScript (200u/μL) with RNase inhibitor

2 x 25μL

2 x 100μL

5x UltraScript Buffer

1 x 200μL

4 x 200μL

UltraScript® Reverse Transcriptase (2500 U/μL)

Component

50 000 Units

UltraScript Reverse Transcriptase (2500 U/μL) with RNase Inhibitor

1 x 20 μL

Glycerol-Free Enzyme Dilution Buffer

1 x 1.5 mL

UltraScript® Reverse Transcriptase (8000 U/μL)

Component

80 000 Units

UltraScript Reverse Transcriptase (8000 U/μL)

1 x 10 μL

Glycerol-Free Enzyme Dilution Buffer

1 x 1.5mL

UltraScript® cDNA Synthesis Kit

Component

20x UltraScript for cDNA Synthesis

5x cDNA Synthesis Mix



25 Reactions

1 x 25 μL

1 x 100 μL

100 Reactions

1 x 100 μL

4 x 100 μL

UltraScript® cDNA Synthesis Kit Separate Oligos

Component

20x UltraScript for cDNA Synthesis

5x UltraScript Buffer

100 μΜ Anchored Oligo(dT)18

100 μM Random Hexamers



25 Reactions

1 x 25 μL

1 x 200 μL

1 x 100 μL

1 x 100 μL

100 Reactions

1 x 100 μL

2 x 200 μL

1 x 100 μL

1 x 100 μL

UltraScript® Reverse Transcriptase

Component

UltraScript (200u/μL) with RNase inhibitor

5x UltraScript Buffer



10,000 Units

2 x 25μL

1 x 200μL

40,000 Units

2 x 100μL

4 x 200μL

UltraScript® Reverse Transcriptase (2500 U/μL)

Component

UltraScript Reverse Transcriptase (2500 U/μL) with RNase Inhibitor

Glycerol-Free Enzyme Dilution Buffer



50 000 Units

1 x 20 μL

1 x 1.5 mL

UltraScript® Reverse Transcriptase (8000 U/μL)

Component

UltraScript Reverse Transcriptase (8000 U/μL)

Glycerol-Free Enzyme Dilution Buffer



80 000 Units

1 x 10 μL

1 x 1.5mL

Reaction Volume

Storage

Not applicable

On arrival, products should be stored between -30 and -15°C. If stored correctly the kit will retain full activity for 12 months.

Reaction Volume

Not applicable

Storage

On arrival, products should be stored between -30 and -15°C. If stored correctly the kit will retain full activity for 12 months.

FAQs

Can long targets (15-20 kb) be reverse transcribed by UltraScript™ RTase?

20 kb RNA reverse transcription can be achieved with all our RTases because RTases jump on and off their primed substrates. Secondary structures, unspecific binding and mis-priming can be the most common causes for the reaction ending prematurely.

We advise using specific primers or oligo(dT) and optimising each aspect of the reaction, for example, the different enzyme concentrations, primers, primer concentration, temperature and temperature cycling and time. Also, the subsequent PCR/qPCR step might need optimisation.

We recommend UltraScript™ 2.0 RTase for reverse transcribing long templates because it can sustain higher temperatures for longer, which may be necessary as the probability of secondary structures in RNA increases with its length.

We’ve included the following paper for your reference1.

1  Thiel, V. et al. Effective amplification of 20-kb DNA by reverse transcription PCR. Anal Biochem 252, 62-70, doi:10.1006/abio.1997.2307 (1997).

Can miRNA be amplified using UltraScript™ cDNA Synthesis Kit?

miRNA should not be amplified using UltraScript™ cDNA Synthesis Kit. UltraScript™ cDNA Synthesis Kit Separate Oligos, UltraScript Reverse Transcriptase, or UltraScript 2.0 Reverse Transcriptase should be used because they come with a buffer that does not contain random hexamers and oligod(T)s, which could interfere with miRNA specific primers.

Can miRNA be amplified using UltraScript™ Reverse Transcriptase?

All our RTases are can be used for miRNA quantification and analysis. However, we do not sell any dedicated kits.

We advise that you use one of the two following approaches:

  • Use universal RT primers and add poly(A) or poly(U) tails (e.g. by poly(U)-polymerase), followed by cDNA synthesis using universal primers1,2.
  • Use specific RT primers and omit the tailing step1,3-5.

If you are unfamiliar with the specifics of those approaches, please refer to the reference list below, which serve as a guideline.

1  Dave, V. P. et al. MicroRNA amplification and detection technologies: opportunities and challenges for point of care diagnostics. Lab Invest 99, 452-469, doi:10.1038/s41374-018-0143-3 (2019).

2  Mei, Q. et al. A facile and specific assay for quantifying microRNA by an optimized RT-qPCR approach. PLoS One 7, e46890, doi:10.1371/journal.pone.0046890 (2012).

3  Chen, C. et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33, e179, doi:10.1093/nar/gni178 (2005).

4  Raymond, C. K., Roberts, B. S., Garrett-Engele, P., Lim, L. P. & Johnson, J. M. Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. RNA 11, 1737-1744, doi:10.1261/rna.2148705 (2005).

5  Androvic, P., Valihrach, L., Elling, J., Sjoback, R. & Kubista, M. Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification. Nucleic Acids Res 45, e144, doi:10.1093/nar/gkx588 (2017).

Can UltraScript™ cDNA Synthesis Kit be used for endpoint and qPCR?

All our RTases and kits can be used for both endpoint and qPCR applications.

Can UltraScript™ cDNA Synthesis Kit reverse transcribe eukaryotic and prokaryotic RNA?

UltraScript™ DNA Synthesis Kit can amplify both eukaryotic and prokaryotic RNA. Our mix contains random hexamers that are universal primers for all types of RNA, provided it contains sufficiently long fragments. Due to the presence of hexamers, RNA from all organisms can be reverse transcribed if it is available for the RTase. There are also oligo-d(T) included in the mix, which work with A-tailed RNA such as eukaryotic mRNA.

For a more selective kit use UltraScript™ Reverse Transcriptase or UltraScript™ 2.0 Reverse Transcriptase as the buffer for these products come without any hexamers and oligo(dT)s. This allows you to determine your own priming strategy.

Can UltraScript™ Reverse Transcriptase be used with blood or plasma samples?

Yes, however we recommend comparing it against UltraScript™ 2.0 for your applications since it is better-suited to handle the various inhibitors present in blood. We also recommend optimisation by performing a titration of the amount of blood/plasma sample for RT reaction.

Can UltraScript™ Reverse Transcriptase do template switching?

All our RTases can be used in template switching applications. We currently do not provide a full protocol or dedicated kit but we recommend referring to this list of articles for reference1-4.

1  Takada, S. & Mano, H. Profiling of microRNA expression by mRAP. Nat Protoc 2, 3136-3145, doi:10.1038/nprot.2007.457 (2007).

2  Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171-181, doi:10.1038/nprot.2014.006 (2014).

3  Moldovan, N. et al. Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus. Front Microbiol 8, 2708, doi:10.3389/fmicb.2017.02708 (2017).

4  Zajac, P., Islam, S., Hochgerner, H., Lonnerberg, P. & Linnarsson, S. Base preferences in non-templated nucleotide incorporation by MMLV-derived reverse transcriptases. PLoS One 8, e85270, doi:10.1371/journal.pone.0085270 (2013).

Is a thermocycler required to perform reverse transcription?

Reverse transcription is an isothermal process so any instrument that can maintain stable temperature will be fine. We do not advise using water baths due to contamination. A thermocycler has the added advantage of a heated lid that circumvents the problem of tube cap condensation.

What are the main differences between UltraScript™ and UltraScript™ 2.0 Reverse Transcriptases?

Both products are derived from wild-type MMLV reverse transcriptase and contain multiple mutations that enhance their function. They are each excellent RTases in their own right. UltraScript™ 2.0 is more thermostable up to 65 °C and above, resistant to inhibitors and capable of reverse transcribing large quantities of RNA up to 3.5 μg. UltraScript™ 2.0 can be also used for applications requiring sensitivity due to a low amount of RNA present, however, this requires substantial optimisation on the users side using the current formulation and we recommend using UltraScript™ instead. UltraScript™ can be used with 1-step kits whereas UltraScript™ 2.0 should be used only as a part of the 2-step process (RT then PCR). UltraScript™ 2.0 is especially recommended for long and difficult templates that contain large amounts secondary structure. Both RTases can be used with cDNA synthesis kit with/or without hexamers and oligo-d(T).

What is the dynamic range of UltraScript™ Reverse Transcriptase?

UltraScript™ RTase can transcribe as little as 4 pg and up to 0.4 μg of RNA. The performance depends on the type of RNA, RNA quality and whether specific primers, oligod(Ts) or random hexamers are used. For higher capacity applications please use UltraScript™ 2.0 Reverse Transcriptase and cDNA Synthesis Kits.

What is the error rate of UltraScript™ Reverse Transcriptase?

Like the great majority of the RTases available on the market UltraScript™ Reverse Transcriptase, as well as all other RTases we offer, are derived from wild-type Moloney Murine Leukemia Virus (MMLV) RTase and have an error rate of 1×10-4 errors/bp1. This also applies to RTases derived from Avian Myeloblastosis Virus (AMV).

Running the reaction at a higher temperature increases fidelity because it destabilises mismatched base pairs2. If low fidelity is required adding manganese will make RTases highly mutagenic and could increase their speed3.

1  Yasukawa, K. et al. Next-generation sequencing-based analysis of reverse transcriptase fidelity. Biochem Biophys Res Commun 492, 147-153, doi:10.1016/j.bbrc.2017.07.169 (2017).

2  Malboeuf, C. M., Isaacs, S. J., Tran, N. H. & Kim, B. Thermal effects on reverse transcription: improvement of accuracy and processivity in cDNA synthesis. Biotechniques 30, 1074-1078, 1080, 1082, passim, doi:10.2144/01305rr06 (2001).

3  Cadwell, R. C. & Joyce, G. F. Randomization of genes by PCR mutagenesis. PCR Methods Appl 2, 28-33 (1992).

What is the maximum length of product possible using UltraScript™ cDNA Synthesis Kit?

The RTase included in UltraScript™ cDNA Synthesis Kit can transcribe very long targets of up to 20 kB with sufficient optimisation1. However, the kit contains random hexamers that will align in random places along the sequence and this will greatly reduce the maximum length of a continuous product. Most of the products will be discontinuous to a large extent nonetheless, the whole sequence of RNA will be covered and subsequently be quantifiable with qPCR. This applies to any cDNA synthesis kit that incorporates random hexamers. If full-length continuous products are required, we recommend UltraScript™ Reverse Transcriptase or UltraScript™ 2.0 Reverse Transcriptase as these products come with a buffer without any hexamers and oligo(dT)s.

1  Thiel, V. et al. Effective amplification of 20 kb DNA by reverse transcription PCR. Anal Biochem 252, 62-70, doi:10.1006/abio.1997.2307 (1997).

What troubleshooting is there for low cDNA yield?

There could be multiple reasons for low yield occurring:

  • The amount or the integrity of your RNA could be insufficient to reach a significant amount of product required for the subsequent endpoint or qPCR step. Try increasing the amount RNA and/or check the quality of your RNA using gel electrophoresis and RIN value. Consider an alternative purification protocol for your RNA and/or use RNase inhibitor early on. All our RTases contain RNase inhibitor but for some applications it might be necessary to add it before RT step
  • You RNA prep may contain inhibitors such as haem, high concentrations of NaCl, SDS, guanidine thiocyanate, melanin, or calcium1. Try an alternative method of purification that gets rid of all substances that can inhibit reaction. You could also try diluting your RNA before the reaction. This will decrease the theoretical yield, but it will also dilute inhibitors to levels more permissible for the RTase to work efficiently. This could still provide enough product for subsequent steps.
  • Some RNAs are inherently difficult to transcribe due to secondary structures and/or stretches of sequences that are not an ideal substrate for RTase. Try increasing the temperature and/or performing RNA denaturation/primer annealing, at 70°C for 10 minutes, before adding RTase.
  • Using RNAseH treatment after RT step can increase the yield, especially for GC rich targets because RNA-DNA heteroduplexes are more stable than DNA-DNA duplexes.
  • If your sequences are still underrepresented it might be necessary to use a more targeted approach with specific primers or just oligo(dT)s. We recommend UltraScript Reverse Transcriptase or UltraScript 2.0 Reverse Transcriptase that come with a buffer without any hexamers and oligo(dT)s for that purpose.

1  Schrader, C., Schielke, A., Ellerbroek, L. & Johne, R. PCR inhibitors – occurrence, properties and removal. J Appl Microbiol 113, 1014-1026, doi:10.1111/j.1365-2672.2012.05384.x (2012).

What troubleshooting is there if smears or non-specific products are visible after using UltraScript™ RTase?

There could be multiple reasons for the appearance of smears or non-specific products after the RT reaction. The following points can be considered for troubleshooting:

  • It’s important to establish if the problem relates to the reverse transcription step or PCR/qPCR step, provided the PCR/qPCR step is performed downstream from the reverse transcription step. Use proper controls and troubleshoot the PCR/qPCR reaction to exclude this from the list of potential steps causing the above problems.
  • Smearing can indicate primer oligomerisation. Oligomerisation can be caused if the oligomers are badly designed and if the template concentration is low. This will result in reverse transcription of primer-dimers or off-target sequences. We advise redesigning primers, increasing reaction temperature, increasing the amount of template, decreasing primer concentration or shortening the reaction time. These considerations can also be used for non-specific bands.
  • Primers may not be specific to your target sequence and may require redesign.
  • Primers against highly repetitive sequences can cause smearing and redesigning your primers may have to be considered.
  • Your RNA may be degraded. Try increasing the amount of RNA and/or check the quality of your RNA using gel electrophoresis and RIN value. Try an alternative purification protocol for your RNA and/or use RNase inhibitor early on. All our RTases contain RNA inhibitor but for some applications it might be necessary to add it before the RT step.
  • Consider the standard precautions when handling RNA such as using gloves, positive displacement pipettes with aerosol barrier, etc.
  • Ensure there is no DNA contamination.

Why is there is a false positive in the RT-qPCR analysis?

Your RNA sample might be contaminated with DNA. We suggest re-purifying your RNA using a better protocol or use thermolabile DNase to get rid of any contamination.