tRNA epitranscriptomic modification mapping answers two questions at once: which modifications are present and at what level, and exactly where they sit on each tRNA. Arraystar's solution combines base-resolution tRNA modification sequencing — m7G TRAC-Seq, m3C HAC-Seq, tRNA Modification Seq (m1A, m3C, m1G, m2,2G), and tRNA Charging Seq — with full-spectrum LC-MS quantification of 55 nucleoside modifications, for quantitative, position-resolved tRNA modification profiling.
Two complementary technical routes for quantitative tRNA modification mapping
tRNAs undergo by far the greatest number of, and the most chemically diverse, post-transcriptional modifications. These modifications are critical for all core aspects of tRNA function, such as folding, stability, and decoding [1]. Defects in tRNA modifications and modification enzymes are linked with human diseases such as cancers, diabetes, neurological syndromes, cardiac conditions, and mitochondrial-linked disorders [3]. Studying tRNA modification is perhaps just as important as tRNA expression profiling.
Reliable modification mapping needs two complementary layers of evidence. Base-resolution sequencing localizes individual modifications on specific tRNAs: m7G TRAC-Seq uses reductive cleavage at m7G; m3C HAC-Seq uses hydrazine-aniline cleavage at m3C; tRNA Modification Seq compares demethylase-treated and untreated tRNAs to identify m1A, m3C, m1G, and m2,2G sites; and tRNA Charging Seq detects modification-induced misincorporation during reverse transcription and simultaneously profiles tRNA expression and charging. Full-spectrum quantification (LC-MS) hydrolyzes tRNAs into single nucleosides and simultaneously profiles 55 nucleoside modifications from total RNA, delivering the global modification profile of the sample.
tRNA epitranscriptomic modification mapping identifies and quantifies post-transcriptional modifications on transfer RNAs, either globally as nucleoside profiles (LC-MS) or at precise base positions on individual tRNAs (TRAC-Seq, HAC-Seq, Modification Seq, tRNA Charging Seq), linking modification status to tRNA function and disease.
Five tRNA modification mapping services — combine them for integrated epitranscriptomic studies
| Service Name | Modification Targets | Resolution | Price |
|---|---|---|---|
| m7G TRAC-Seq | m7G | Single-nucleotide | |
| m3C HAC-Seq | m3C | Single-nucleotide | |
| tRNA Modification Seq – m1A, m3C, m1G, m2,2G | m1A, m3C, m1G, m2,2G | Single-base | |
| tRNA Charging Seq | Broad modification coverage (e.g. m1A, m1G, m3C, acp3U) + tRNA expression & charging | Single-nucleotide (predicted) | |
| LC-MS Based tRNA Modification Analysis | 55 nucleoside modifications (global profile) | Nucleoside-level quantification |
Why researchers choose Arraystar for tRNA modification mapping
LC-MS simultaneously profiles 55 nucleoside modifications from total RNA, while sequencing methods map individual modifications at single-base resolution on specific tRNAs.
TRAC-Seq and HAC-Seq rely on highly specific chemical reactions rather than antibody affinity, eliminating background from non-specific binding and enabling quantitative stoichiometry assessment.
tRNA Modification Seq simultaneously detects and quantifies m1A, m3C, m1G, and m2,2G modifications at single-base resolution by comparing demethylase-treated and untreated tRNAs.
tRNA Charging Seq profiles tRNA expression, tRNA modifications, and tRNA charging at once, with broad modification coverage (e.g. m1A, m1G, m3C, acp3U) predicted at single-nucleotide resolution.
Full-service workflows from sample QC through tRNA isolation, library preparation or nucleoside analysis, sequencing or LC-MS/MS acquisition, to comprehensive bioinformatics and report.
All services work well together with Arraystar's broad tRNA research technologies — tRNA-seq, tRF&tiRNA-Seq, PCR arrays, and rtStar™ pretreatment kits — for integrated studies.
tRNA modifications — chemically diverse marks that control tRNA function and disease
tRNAs are the fundamental component of mRNA decoding and protein translation. tRNAs undergo by far the greatest number of and the most chemically diverse post-transcriptional modifications, which are critical for all core aspects of tRNA function, such as folding, stability, and decoding [1]. Typically, modifications in the main body of tRNA are crucial for tRNA structure folding, stability, rigidity, and flexibility, whereas modifications in the anticodon loop affect decoding by open loop structure, codon-anticodon pairing, wobbling, and preventing translational frameshifts. Additionally, modified nucleosides serve as identity determinants for aminoacyl-tRNA synthetase for extra amino acid recognition accuracy [2]. In general, hypomodified tRNAs are targeted for degradation.
Defects in tRNA modifications and modification enzymes are linked with human diseases such as cancers, diabetes, neurological syndromes, cardiac conditions, and mitochondrial-linked disorders (Fig. 1) [3]. m3C at position 32 of the anticodon loop maintains tRNA structure, decoding accuracy, and translation efficiency; dysregulation of m3C-related enzymes and tRNA m3C modifications has been associated with tumor progression and metastasis in cancers such as hepatocellular carcinoma and breast cancer, as well as neurological and mitochondrial disorders. m7G at position 46 in the variable loop, deposited by the METTL1/WDR4 complex, maintains tRNA structural integrity and enhances translation of codon-enriched mRNAs; METTL1 overexpression drives tumorigenesis in leukemia, glioblastoma, cholangiocarcinoma, and lung cancer. Beyond abundance, tRNA expression and charging — the aminoacylation state that determines translation activity — complete the functional picture that epitranscriptomic mapping must resolve (Fig. 2).
From total RNA to a quantitative, position-resolved tRNA modification map
Total RNA quality and quantity assessment before the project proceeds, with small-RNA-retaining purification.
LC-MS route: tRNA isolation from total RNA, complete hydrolysis and dephosphorylation to single nucleosides, and UHPLC-MS/MS quantification of 55 nucleoside modifications.
Select the sequencing method by target: m7G TRAC-Seq (reductive cleavage), m3C HAC-Seq (hydrazine-aniline cleavage), tRNA Modification Seq (demethylase ± comparison), or tRNA Charging Seq (reverse-transcription misincorporation).
Differential modification analysis, IGV read alignments for site identification, sequence motif analysis, and volcano plots.
Combine global and positional modification data, and cross-reference with tRNA expression, charging, and tRF/tiRNA profiles.
Detailed bioinformatics analyses included in every tRNA modification mapping service
Each service includes detailed bioinformatics analyses to facilitate insights into tRNA modifications in biology, diseases, and biomarker applications. m7G TRAC-Seq delivers m7G sites and modification levels by cleavage scores; m3C HAC-Seq identifies m3C sites at single-nucleotide resolution by cleavage ratio; tRNA Modification Seq analyzes modification sites, methylation levels, and tRNA expression at once; and tRNA Charging Seq provides a wealth of tRNA multi-omics data with common analyses (e.g. differential analyses) and detailed annotations. LC-MS projects report raw and normalized peak data, Total Ion Current chromatograms of nucleosides, and differential modification of nucleosides among samples.
tRNA modification mapping across biology and disease
m7G-modified tRNAs (e.g. Arg-TCT-4-1, Lys-CTT, Val-AAC) drive tumorigenesis in leukemia, glioblastoma, cholangiocarcinoma, and lung cancer (Mol Cell, 2021); m3C dysregulation is associated with tumor progression and metastasis in hepatocellular carcinoma and breast cancer.
METTL8-dependent mitochondrial tRNA m3C is indispensable for neural stem cell maintenance (Cell Stem Cell, 2023); DALRD3-dependent modification of tRNA-Arg is crucial for neurological function (Nat Commun, 2020).
m3C32 tRNA modification controls serine codon-biased mRNA translation, cell cycle, and DNA-damage response (Nat Commun, 2024); m7G-modified tRNAs enhance translation efficiency of codon-enriched oncogenic mRNAs.
METTL1/WDR4-mediated m7G tRNA methylome is required for embryonic stem cell self-renewal and differentiation (Mol Cell, 2018); METTL6-mediated modification of tRNA-Ser supports pluripotency and tumorigenesis (Sci Adv, 2020).
tRNA modification profiles link to diabetes and cardiac conditions; glutamyl-prolyl-tRNA synthetase regulates pro-fibrotic protein synthesis during cardiac fibrosis (Circ Res, 2020); tRNA Charging Seq applications include cardiac fibrosis and cancer drug resistance.
Valine aminoacyl-tRNA synthetase promotes therapy resistance in melanoma (Nat Cell Biol, 2024); dual targeting of m7G tRNA modification and histone acetylation evokes osteosarcoma chemosensitization (Adv Mater, 2025).
Method selection depends on your modification targets and the resolution you need
| Dimension | TRAC-Seq (m7G) | HAC-Seq (m3C) | tRNA Modification Seq | tRNA Charging Seq | LC-MS Based tRNA Modification Analysis |
|---|---|---|---|---|---|
| Modification targets | m7G | m3C | m1A, m3C, m1G, m2,2G | Broad coverage (e.g. m1A, m1G, m3C, acp3U), predicted | 55 nucleoside modifications (global profile) |
| Resolution | Single-nucleotide | Single-nucleotide | Single-base | Single-nucleotide (predicted) | Nucleoside-level quantification |
| Key principle | NaBH4/aniline reductive cleavage | Hydrazine-aniline cleavage | Demethylase-treated comparison (methylation index) | Reverse transcription misincorporation | Hydrolysis + LC-MS/MS of single nucleosides |
| Additional profiles | m7G sites & levels | m3C sites & levels | tRNA expression | tRNA expression & charging | Global nucleoside modification status |
| Recommended total RNA | > 5 µg* | > 5 µg* | > 5 µg* | > 5 µg* | 10*–15 µg |
Official Arraystar sample submission requirements for tRNA modification mapping projects
| Service / Sample Type | Requirement | Notes |
|---|---|---|
| TRAC-Seq / HAC-Seq / tRNA Modification Seq | > 5 µg total RNA* | Recommended minimum for the entire experiment including sample QC |
| tRNA Charging Seq | > 5 µg total RNA* | Recommended minimum for the entire experiment including sample QC |
| LC-MS tRNA Modification Analysis | 10*–15 µg total RNA | tRNA isolation performed from the submitted total RNA |
| Cultured cells | 2 × 10⁶ cells | Lyse immediately in TRIzol (1 mL per 5–10 × 10⁶ suspension cells or 10 cm² dish); ship on dry ice |
| Tissue | 10–25 mg | RNAlater (≥10 volumes, 2–8 °C overnight), TRIzol (1 mL per 10–25 mg), or fresh-frozen in liquid nitrogen |
| Whole blood | 2–3 mL (0.3 mL/aliquot) | Services with pretreatment → use the “With IP or Pretreatment” volumes; ship on dry ice |
| Plasma / Serum | 2–5 mL (0.3 mL/aliquot) | EDTA or citrate anticoagulant; do NOT use heparin; ship on dry ice |
* Recommended minimum amount per sample for the entire experiment in a single attempt, including sample QC; supply twice the recommended minimum to avoid project delays. If the minimum amount is not obtainable, contact support@arraystar.com for special arrangements. Arraystar performs sample QC upon receipt; QC assessment is final. Low amount/quality samples may proceed with consent — data quality and success rate may decline. Shipping address: ATTN: Samples Receiving (Project#______), Arraystar Inc., 9430 Key West Avenue #128, Rockville, MD 20850 USA, Tel: 888-416-6343.
Common questions about tRNA epitranscriptomic modification mapping
Featured Client Publications in tRNA Modification Research
Arraystar combines full-spectrum LC-MS quantification of 55 nucleoside modifications with single-base resolution tRNA modification sequencing — get a quote and a project design tailored to your modification targets.