Product Class: Other

Induro® Reverse Transcriptase

Experience a new class of performance with a group II intron-encoded RT.


Catalog #M0681

Product Introduction

Product Information

Description

Induro Reverse Transcriptase is a group II intron-encoded RT that exhibits high processivity, increased thermostability, and increased tolerance of inhibitors in the synthesis of cDNA from RNA. It is an ideal enzyme for challenging cDNA synthesis from long transcripts, RNAs with strong secondary structures, and RNA samples with inhibitors. With improved 5' sequencing coverage of long transcripts, Induro Reverse Transcriptase can enable RNA-seq applications such as direct RNA and long read cDNA sequencing workflows.

Figure 1: Induro Reverse Transcriptase exhibits high processivity, permitting rapid cDNA synthesis



Induro Reverse Transcriptase can synthesize a full-length 12 kb cDNA product within 5 min. at 55°C. In vitro transcribed poly(A) RNA templates (1 kb, 4 kb, 8 kb or 12 kb) were used to investigate full-length cDNA synthesis. After first-strand cDNA synthesis, RNA was hydrolyzed immediately by NaOH. Subsequently, an aliquot of the cDNA products was used to make full-length ds cDNA in the presence of a 5´ specific primer. Equal volume of ds cDNA was analyzed on an agarose gel.



Figure 2: Thermostability of Induro Reverse Transcriptase allows robust synthesis at higher reaction temperatures



Induro Reverse Transcriptase can make a full-length 12 kb cDNA product in 10 min. using incubation temperatures of 45°C to 60°C. In vitro transcribed poly(A) RNA templates (1 kb, 4 kb, 8 kb or 12 kb) were used to investigate full-length cDNA synthesis. After first strand cDNA synthesis, RNA was hydrolyzed immediately by NaOH. Subsequently, an aliquot of the cDNA products was used to make full-length ds cDNA in the presence of a 5´ specific primer. Equal volume of ds cDNA was analyzed on an agarose gel.



Figure 3: Induro Reverse Transcriptase generates highest yields of long cDNA



Induro Reverse Transcriptase generates the highest product yields for cDNA ≥ 8 kb. RNA templates were in vitro transcribed poly(A) RNA (1 kb, 4 kb, 8 kb, 10 kb or 12 kb). After first strand cDNA synthesis, RNA was degraded and the second strand cDNA synthesis was performed in the presence of a 5´ specific primer.



Figure 4: Induro Reverse Transcriptase enables long cDNA synthesis from complex human RNA

 

Induro Reverse Transcriptase can synthesize products greater than 14 kb from human total RNA using a 10-minute incubation at 50°C, 55°C or 60°C. After first strand cDNA synthesis, an aliquot was amplified by PCR using LongAmp® Taq 2X Master Mix (NEB #M0287).



Figure 5: Induro Reverse Transcriptase is more inhibitor-resistant than TGIRT



Induro Reverse Transcriptase (I) shows a higher tolerance to inhibitors commonly present in RNA biological samples than TGIRT®-III Enzyme (T). 1 μg of in vitro transcribed 8 kb poly(A) RNA was used in a 20 μl cDNA synthesis reaction with oligo dT23VN. All reaction components including the RT enzymes were mixed well on ice before the inhibitor was added. The cDNA synthesis condition for Induro was 55°C, 10 min. followed by 95°C, 1 minute. The cDNA synthesis condition for TGIRT was 60°C, 60 min. followed by 95°C, 1 min. The amounts of inhibitors were the final concentration in the first strand cDNA synthesis. The second-strand cDNA synthesis was performed in the presence of a 5´ specific primer.



Figure 6: Induro Reverse Transcriptase produces cDNA libraries with longer transcripts

Long Read

Libraries of cDNA were prepared using either Induro RT or Maxima™ RT from total mouse brain RNA before sequencing on an Oxford Nanopore® GridION®. 3.25M reads were sampled from each library (seqtk sample) and aligned to the GRCm39 transcriptome (minimap 2.17). Average 5′→3′ coverage was plotted for each transcript length group with the top 50% of transcripts shown in a heatmap (yellow = high coverage). Reads mapping with mapQ ≥ 1 to either a 3.5 kb transcript (pelkhb2) (A), or a 8.2 kb transcript (adgrl1) (B) are shown in IGV (2.14.0) with Induro and Maxima RT. Induro libraries contain longer transcripts across all transcript length groups.


 
This product is related to the following categories:
cDNA Synthesis & Reverse Transcriptases,
This product can be used in the following applications:
RT-PCR,
cDNA Synthesis,
Reverse Transcription (cDNA Synthesis),
RT-PCR & cDNA Synthesis

Properties & Usage

Protocols, Manuals & Usage

Protocols

  1. Induro® Reverse Transcriptase Protocol (NEB #M0681)

FAQs & Troubleshooting

FAQs

  1. What is the difference between Induro Reverse Transcriptase (NEB #M0681) and ProtoScript II Reverse Transcriptase (NEB #M0368)?
  2. How do I choose between Induro Reverse Transcriptase and the different LunaScript and ProtoScript products?
  3. Can Induro Reverse Transcriptase be used in Oxford Nanopore Technologies® direct RNA sequencing?
  4. What is the optimal reaction temperature for Induro Reverse Transcriptase?
  5. Is it possible to inactivate Induro Reverse Transcriptase?
  6. What is the recommended incubation time for Induro Reverse Transcriptase?
  7. What is the longest cDNA generated by Induro Reverse Transcriptase?
  8. How much Induro Reverse Transcriptase should be used in a 20 µl reaction? Can the yield be further improved by using more enzyme?
  9. What RNA samples are compatible with Induro Reverse Transcriptase?
  10. How much RNA template should I use?
  11. What primer should I use with Induro Reverse Transcriptase? Can I spike in gene-specific primers to the cDNA reaction?
  12. What applications are best for Induro Reverse Transcriptase?
  13. Can the cDNA products generated by Induro Reverse Transcriptase be used in real-time PCR analysis?
  14. What thermostable DNA polymerase can be used for PCR after cDNA synthesis?
  15. Why do I have low cDNA yields?
  16. Should I include a No-RT control reaction?
  17. How do I know whether my template RNA is of good quality?
  18. Can I store Induro Reverse Transcriptase and its reaction buffer at 4°C?
  19. Can I set up my cDNA synthesis reaction at room temperature when using Induro Reverse Transcriptase?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]


Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email [email protected].

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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