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Small RNA Modification Sequencing

tRNA Modification Seq — m1A, m3C, m1G, m2,2G — Simultaneous Multi-Methylation Profiling at Single-Base Resolution

tRNAs modified with multiple methylations (m1A, m3C, m1G, m2,2G) can be simultaneously and quantitatively profiled at single-base resolution by tRNA Modification Seq – m1A/m3C/m1G/m2,2G. These modifications disrupt Watson–Crick base pairing and can induce mismatch mutations due to misincorporation by reverse transcription.

Four methylation types in one assay · single-base resolution · tRNA expression included

Overview

Demethylase-comparison tRNA-seq for quantitative methylation profiling

To identify modification sites and quantify the methylation in a tRNA, the highly optimized tRNA-seq is performed on the tRNA with and without demethylase treatment. The modification is detected by methylation induced mutation index (MI) at each base position compared with its demethylase-treated tRNA.

Among m1A, m3C, m1G, and m2,2G modifications, the m1G and m2,2G are further distinguished by their known distinct base positions in mammalian tRNAs (m1G at 9 and 37; m2,2G at 26 tRNA base positions). Bioinformatic analyses and annotations are included in the tRNA Modification Seq – m1A/m3C/m1G/m2,2G service.

What is tRNA Modification Seq?

tRNA Modification Seq profiles m1A, m3C, m1G, and m2,2G methylation in tRNAs simultaneously at single-base resolution. Highly optimized tRNA-seq is performed on tRNA with and without demethylase treatment; methylation is quantified by the methylation induced mutation index at each base position.

Service at a Glance

Standard tRNA Modification Seq project — custom designs and add-on analyses available on request

Service NamePrice
tRNA Modification Seq - m1A, m3C, m1G, m2,2G

Benefits

Why researchers choose Arraystar tRNA Modification Seq

🧬

Multiple Methylation Detection

Simultaneously profile m1A, m3C, m1G, and m2,2G modifications in tRNAs.

⚗️

High-Efficiency Methylation Removal

Efficiently remove internal and terminal modifications in the demethylase-treated tRNA using the Arraystar rtStar™ tRNA Pretreatment Kit.

🎯

Single-Base Resolution

Identify the exact sites of m1A, m3C, m1G, and m2,2G methylation modifications by comparing demethylase-untreated tRNA with demethylase-treated tRNA side by side.

📊

Comprehensive tRNA Epitranscriptomic Analysis

Analyze modification sites, methylation levels, and tRNA expression all at once.

🚀

High-Performance tRNA Sequencing

Highly optimized tRNA-seq with a rigorous QC process, overcoming difficulties of regular sequencing for tRNA.

🔗

Integrated tRNA Solutions

Works well together with our broad portfolio of tRNA research technologies: tRNA PCR array, LC-MS based tRNA modification analysis, tRF&tiRNA-seq, and tRF&tiRNA PCR array.

Background

How m1A, m3C, m1G, and m2,2G are detected in tRNAs

tRNAs modified with multiple methylations (m1A, m3C, m1G, m2,2G) can be simultaneously and quantitatively profiled at single-base resolution by tRNA Modification Seq – m1A/m3C/m1G/m2,2G. These modifications disrupt Watson–Crick base pairing and can induce mismatch mutations due to misincorporation by reverse transcription.

To identify modification sites and quantify the methylation in a tRNA, the highly optimized tRNA-seq is performed on the tRNA with and without demethylase treatment. The modification is detected by methylation induced mutation index (MI) at each base position compared with its demethylase-treated tRNA. Among m1A, m3C, m1G, and m2,2G modifications, the m1G and m2,2G are further distinguished by their known distinct base positions in mammalian tRNAs (m1G at 9 and 37; m2,2G at 26 tRNA base positions).

Figure 1. Workflow of tRNA Modification Seq. m1A, m3C, m1G, and m2,2G modifications are identified by their induced misincorporation mutations in reverse transcription compared with demethylase-treated tRNA.
Figure 1. Workflow of tRNA Modification Seq. m1A, m3C, m1G, and m2,2G modifications are identified by their induced misincorporation mutations in reverse transcription compared with demethylase-treated tRNA.

tRNA Modification Seq Workflow

From total RNA to single-base methylation maps for four modification types

1

Sample QC

RNA quality and quantity assessment before the project proceeds, with small-RNA-retaining purification.

2

Demethylase ± Treatment

Each tRNA sample is processed with and without demethylase treatment to generate the paired comparison required for methylation detection.

3

rtStar™ Pretreatment & Library Construction

Internal and terminal tRNA modifications are efficiently removed with the Arraystar rtStar™ tRNA Pretreatment Kit, enabling highly optimized tRNA-seq.

4

High-Throughput Sequencing

High-throughput sequencing of the demethylase-treated and untreated tRNA libraries.

5

Bioinformatics

Methylation induced mutation index (MI) computed at each base position; modification sites, methylation levels, and tRNA expression analyzed with volcano plots and detailed annotations.

Bioinformatics & Deliverables

Bioinformatic analyses and annotations included in every project

Bioinformatic analyses and annotations are included in tRNA Modification Seq – m1A/m3C/m1G/m2,2G Service.

Standard Deliverables

Figure 2. A Volcano plot showing differential tRNA methylation sites between two comparison groups.
Figure 2. A Volcano plot showing differential tRNA methylation sites between two comparison groups.

Research Applications

Choose the right tool for tRNA modification profiling

Single-Base Modification Mapping

Identify the exact sites of m1A, m3C, m1G, and m2,2G modifications at specific base positions in anticodon or body regions of tRNAs.

Multi-Modification Studies

Simultaneously detect multiple methylation types (m1A/m3C/m1G/m2,2G), for tRNAs that are typically modified by multiple types of modifications.

Epitranscriptomic Research

Combine modification sites, methylation levels, and tRNA expression analysis in one service.

Integrated tRNA Portfolio

Works together with tRNA PCR array, LC-MS based tRNA modification analysis, tRF&tiRNA-seq, and tRF&tiRNA PCR array for integrated tRNA solutions.

Choosing the Right Tool for tRNA Modification Profiling

Arraystar offers both tRNA Modification Seq – m1A/m3C/m1G/m2,2G and Small RNA Modification Microarray for tRNA modification profiling. The comparison chart below can help you choose the most appropriate tool for your research purpose.

DimensiontRNA Modification Seq – m1A/m3C/m1G/m2,2GSmall RNA Modification Microarray
RNA classesFor tRNA onlyFor multiple small RNA classes including miRNAs, pre-miRNAs, tRNAs and tsRNAs (tRFs and tiRNAs).
Modification sitesSingle-base resolution of modification sites, important info for tRNA modifications at specific base positions in anticodon or body regions.For modification detection regardless of modification sites in small size RNAs.
Modification typesSimultaneous detection of multiple methylation types (m1A/m3C/m1G/m2,2G), for tRNAs that are typically modified by multiple types of modifications.One type of modification per project.

Sample Requirements

Official Arraystar sample submission requirements for tRNA Modification Seq

RNA Amount & Quality

  • Total RNA input: > 5 µg per sample (official recommended minimum for the entire experiment in a single attempt, including sample QC). Supply twice the recommended minimum to avoid project delays.
  • Purification: TRIzol / RNA precipitation or an RNA isolation kit. Because tRNA is < 200 nt, use a kit specified to retain small RNAs (e.g. Qiagen miRNeasy).
  • Concentration: > 20 ng/µL by Nanodrop; OD260/280 ~2.0 (acceptable 1.7–2.1); OD260/230 > 1.8.
  • Integrity: sharp 18S/28S rRNA bands by gel, or RIN > 7.0 by Bioanalyzer (serum/plasma/exosome/FFPE RNA exempt).
  • DNase treatment: optional for gDNA removal; required if the sample is also used for qPCR.

Shipping Instructions

  • Ship RNA in nuclease-free water (> 20 ng/µL), freeze-dried, or in ethanol; store at −80 °C or in liquid nitrogen.
  • Use nuclease-free certified, screw-cap 1.5 mL microtubes; seal caps with Parafilm; place tubes in a plastic bag.
  • Use 10 kg dry ice as refrigerant; include a signed Project Form and the sample list.
  • Ship to: Arraystar Inc., 9430 Key West Avenue #128, Rockville, MD 20850, USA. Contact us before shipping.

FAQ

Common questions about tRNA Modification Seq

Which modifications does this service profile?
The service simultaneously and quantitatively profiles four tRNA methylation types: m1A, m3C, m1G, and m2,2G. These modifications disrupt Watson–Crick base pairing and induce mismatch mutations due to misincorporation by reverse transcription, which is the basis of their detection at single-base resolution.
How are modification sites identified?
Highly optimized tRNA-seq is performed on tRNA with and without demethylase treatment. The modification is detected by the methylation induced mutation index (MI) at each base position compared with its demethylase-treated tRNA. m1G and m2,2G are further distinguished by their known distinct base positions in mammalian tRNAs (m1G at 9 and 37; m2,2G at 26).
What is the minimum amount of RNA required?
We recommend more than 5 µg of total RNA per sample — the official Arraystar recommended minimum for the entire experiment in a single attempt, including sample QC. Supplying twice the recommended minimum helps avoid project delays. RNA integrity must be preserved, because degraded RNA cannot be rescued by downstream steps.
How is this different from the Small RNA Modification Microarray?
tRNA Modification Seq is for tRNA only and provides single-base resolution of modification sites, important for tRNA modifications at specific base positions in anticodon or body regions. The Small RNA Modification Microarray covers multiple small RNA classes including miRNAs, pre-miRNAs, tRNAs and tsRNAs, but detects one type of modification per project regardless of sites.
What bioinformatics analyses are included?
Bioinformatic analyses and annotations are included in the service. Projects deliver modification sites at single-base resolution, methylation levels, and tRNA expression analysis, including volcano plots showing differential tRNA methylation sites between two comparison groups. Results are reported with publication-ready graphics and visualization.
Can this service be combined with other tRNA technologies?
Yes. The service works well together with Arraystar's broad portfolio of tRNA research technologies, including the tRNA PCR array, LC-MS based tRNA modification analysis, tRF&tiRNA-seq, and tRF&tiRNA PCR array. This lets you combine modification profiling with expression and quantification approaches in one study.

Selected Publications

Key References for tRNA Modification Sequencing

  1. Cui J, et al. m(3)C32 tRNA modification controls serine codon-biased mRNA translation, cell cycle, and DNA-damage response. Nature Communications, 2024. PMID: 38982125
  2. Lentini JM, et al. DALRD3 encodes a protein mutated in epileptic encephalopathy that targets arginine tRNAs for 3-methylcytosine modification. Nature Communications, 2020. PMID: 32427860
  3. Zhang F, et al. Epitranscriptomic regulation of cortical neurogenesis via Mettl8-dependent mitochondrial tRNA m(3)C modification. Cell Stem Cell, 2023. PMID: 36764294
  4. Orellana EA, Siegal E, Gregory RI. tRNA dysregulation and disease. Nature Reviews Genetics, 2022. PMID: 35681060

Ready to Map tRNA Methylation at Single-Base Resolution?

Arraystar tRNA Modification Seq profiles m1A, m3C, m1G, and m2,2G simultaneously with tRNA expression — get a quote and a project timeline tailored to your study.