RNA 5hmC Modification Profile (hMeRIP-Seq)
Antibody enrichment for 5-hydroxymethylcytosine on RNA, giving a transcript-level profile of where this cytosine oxidation product is enriched.
01What the service reports
RNA 5hmC Modification Profile (hMeRIP-Seq) is an antibody-based enrichment service for 5-hydroxymethylcytosine in RNA. Fragments carrying 5hmC are captured, sequenced and mapped to produce a profile of where the modification is enriched.
The result is a region-level profile. Captured fragments map back to stretches of a transcript, and enrichment relative to an input control describes how much 5hmC signal those regions carry and how the signal differs between samples.
5hmC on RNA is also approached through base-conversion chemistry, which reads the modification differently and resolves it per nucleotide. The antibody route and the conversion route are therefore complementary rather than competing, and the choice follows from whether the study needs a broad comparison or a resolved position. Arraystar runs the enrichment workflow from sample QC to the analyzed profile.
02Benefits
- Broad transcript coverage: the profile surveys 5hmC signal across transcripts rather than at selected positions.
- Suits comparative designs: enrichment data from several samples compare cleanly, which is useful for tissue or treatment series.
- Annotated regions: enriched regions are linked to transcripts, giving a gene-level view of the signal.
- Complementary to conversion chemistry: findings from the profile can be taken to a conversion-based method for position-level detail.
- One service, whole workflow: RNA QC, capture, library construction, sequencing and analysis are delivered together.
| Service Name | RNA Class | Price |
|---|---|---|
| RNA 5hmC Modification Profile (hMeRIP-Seq) | mRNA | |
| RNA 5hmC Modification Profile (hMeRIP-Seq) | mRNA and lncRNA |
03Background and detection principle
5-Hydroxymethylcytosine is an oxidised form of cytosine, and it can be recognized by an antibody raised against it. In this workflow the RNA is fragmented, incubated with that antibody, and the captured fragments are sequenced next to an input control. Regions enriched over input are reported and annotated.
The boundary again comes from capture. Enrichment reports that a fragment carried 5hmC, so the signal is localised to a region rather than to a cytosine, and the profile also reflects how efficiently the antibody bound its target. Read as a comparison between samples, the profile shows where 5hmC signal concentrates and how it moves.
An antibody route reports where 5hmC concentrates on a transcript; base conversion is what reads it one cytosine at a time.Delatte B. et al., Science 2016 · DOI 10.1126/science.aac5253
04Workflow
The service runs in five steps from sample to analyzed 5hmC profile:
- Sample QC, covering integrity and purity before capture.
- Fragmentation of the RNA into a range suitable for antibody binding.
- Immunoprecipitation with an anti-5hmC antibody, against an input control.
- Library construction from the enriched and input fractions.
- Sequencing and analysis to call enriched regions and compare samples.
05Bioinformatics and deliverables
Reads are mapped and enriched regions are called against input, then annotated to transcript features. The analysis set covers region calling and annotation, distribution across features, differential enrichment and genome-browser visualization.
Deliverables
- Raw 5hmC mRNA enrichment sequencing data files (FASTQ), formatted for GEO/SRA submission.
- QC reports and sorted, indexed BAM alignment files with core mapping statistics.
- 5hmC enrichment region tables (XLSX) with genomic coordinates and annotated host genes.
- Region distribution plots over transcript features (PDF/PNG).
- Differential 5hmC enrichment tables (XLSX) between sample groups.
- Genome-browser signal tracks (bigWig/bedGraph) and a structured project report.
06Research applications
- Neuroscience: 5hmC on RNA has been studied in brain tissue, where regional and disease comparisons are a natural design.
- Developmental biology: tissue and stage series can be compared to see where 5hmC signal changes.
- Cancer research: tumour and normal samples can be compared at the transcript level.
- Enzyme and pathway work: samples with altered TET-family activity can be compared to see how the profile responds.
- Planning conversion-based work: regions that stand out in the profile are the candidates for a position-level method.
07Sample requirements
Storage. Cells and tissues: preserve in TRIzol or an RNA stabilisation solution, snap freeze in liquid nitrogen and store at −80 °C. RNA: resuspend in ethanol or RNase-free ultrapure water, store at −80 °C and avoid repeated freeze–thaw cycles.
Shipping. Place the sample in an RNase-free microcentrifuge tube, seal it against leakage, and ship on dry ice with adequate insulation.
| Sample type | Notes |
|---|---|
| Whole blood | EDTA tubes only; heparin is not compatible with the downstream procedure. |
| Cultured cells | Cell pellets are preferred. |
| Tissue | Fresh or frozen specimens; avoid necrotic material. |
| Total RNA | OD 260/280 ≥ 1.8 and RIN ≥ 7, with no visible degradation. Submit at least 200 µg total RNA. |
08FAQ
What if the study needs the position rather than the profile?
Not by this route. hMeRIP-Seq reports where 5hmC is enriched across transcripts. The single-base services cover the marks shown on the modification types page, and 5hmC is not among them, so a position-level 5hmC claim needs a route outside this line. The profile here still answers which transcripts carry the mark and how that changes.
Is hMeRIP-Seq a single-base method for 5hmC?
No. Capture localises the signal to a region of a transcript. Reading 5hmC at individual cytosines is the job of a base-conversion method.
Why is this route described as complementary to conversion chemistry?
Because the two answer different questions. The antibody route gives breadth across transcripts and samples, while conversion chemistry gives position. Using them in sequence is common in 5hmC studies.
How does the antibody affect interpretation?
It determines which fragments are captured, so the profile reflects the antibody’s binding behavior alongside the 5hmC content of the sample. Comparative reading is the reliable use of the output.
Is an input control needed?
Yes. Enrichment calls depend on comparing the captured fraction against input, which is part of the standard workflow.
Which sample types can be submitted?
Total RNA, cultured cells, tissue or blood, subject to the quality thresholds listed on this page.
09References
- Delatte B, Wang F, Ngoc LV, et al. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine. Science 2016;351:282–285. DOI: 10.1126/science.aac5253