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Related Concept Videos

RNA-seq03:21

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...
RACE - Rapid Amplification of cDNA Ends02:35

RACE - Rapid Amplification of cDNA Ends

Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
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One-Step Affinity Purification of MarathonRT Reverse Transcriptase for RNA Sequencing Applications.

Jenni K Pedor1,2, Pavlina Gregorova1,2, Salla M Kalaniemi1

  • 1RNAcious Laboratory, Department of Molecular and Integrative Biosciences, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland.

Bio-Protocol
|June 29, 2026
PubMed
Summary

Researchers developed a simplified protocol for producing highly active MarathonRT (MRT), a next-generation reverse transcriptase crucial for analyzing transfer RNAs (tRNAs). This cost-effective method yields stable, reliable enzyme batches suitable for RNA sequencing applications.

Keywords:
Enzyme purificationMarathonRTNext-generation reverse transcriptaseRNA-seqReverse transcriptiontRNA

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genomics

Background:

  • Transfer RNAs (tRNAs) are vital for translation and cellular processes.
  • High-throughput sequencing methods for tRNA analysis face challenges due to tRNA secondary structures and modifications.
  • Current commercial next-generation reverse transcriptases (ngRTs) are costly and lack alternatives.

Purpose of the Study:

  • To present a simplified, cost-effective protocol for expressing and purifying the next-generation reverse transcriptase MarathonRT (MRT).
  • To develop a reliable assay for measuring MRT enzymatic activity and ensure batch consistency.
  • To validate MRT's performance in RNA sequencing pipelines as an alternative to commercial ngRTs.

Main Methods:

  • Developed a streamlined expression and purification protocol for MRT.
  • Created a simple colorimetric assay for quantifying MRT specific activity.
  • Validated the purified MRT in the mim-tRNAseq pipeline against commercial ngRTs.

Main Results:

  • Achieved a simplified, cost-effective MRT production protocol yielding highly active and stable enzyme.
  • The protocol mitigates previous precipitation issues and reduces purification steps.
  • MRT demonstrated performance comparable to commercial ngRTs (Induro, UltraMarathonRT) in sequencing applications.

Conclusions:

  • The simplified MRT protocol provides a stable, reproducible, and cost-effective alternative for next-generation reverse transcription.
  • The developed assay enables consistent batch quality control for MRT.
  • This work offers a valuable, accessible tool for tRNA analysis and other RNA sequencing applications.