Arraystar m6A Single Nucleotide Arrays based on methyl-sensitive MazF RNase can locate the exact m6A site at single nucleotide resolution and quantify the fraction of modification. With these capabilities unachievable by other currently existing techniques, the arrays will power the research on m6A dynamics, molecular mechanisms, biological functions, and disease associations. In particular, the arrays add invaluable benefits which are lacking for conventional m6A-seq analysis:
Benefits
• An orthogonal methodology for m6A detection. For the first time, the arrays now allow systematic m6A profiling independent of m6A-antibody immunoprecipitation based approaches such as MeRIP or miCLIP. Learn more>>
• Single-Nucleotide resolution for m6A site location. Compared with MeRIP-seq at a resolution limited to 100~200 nt, the arrays based on MazF allow precise detection of m6ACA at single nucleotide resolution. Learn more>>
• m6A modification stoichiometry. The fraction or percentage of m6A modification is quantified at each interrogated site, addressing the unfulfilled long-standing need in determining the dynamic m6A status. Learn more>>
• Tolerant of poor quality RNAs. MazF based enzymatic detection of m6A works with even heavily degraded RNAs, such as RNAs extracted from serum, plasma, exosomes, or FFPE samples. Learn more>>
• Low RNA sample amount requirement. The arrays use as low as 1 ug total RNA, compared with > 100 ug for MeRIP-seq. m6A profiling can now be performed on rare samples, precious pathological specimens, particular histological sites, low yield sorted cells, or small animal models. Learn more>>
• Reliable collection and systematical annotations. Arraystar established a specialized pipeline to collect and annotate the quantifiable m6A sites with Single-ACA, Poly-ACA, or Cluster-ACA. To learn how to collect quantifiable Single-ACA, Poly-ACA, or Cluster-ACA, please click here. To learn the functional significance of these m6A sites, please click here.
Watch Video> m6A Profiling at Single Nucleotide Resolution
RNase MazF has the enzymatic property to cleave single stranded RNA 5’ immediate to unmethylated (ACA) sequence, but not methylated (m6ACA) [5, 10] (Fig. 1). Based on MazF digestion, the RNA fragments with uncleaved m6ACA and the input RNA without MazF digestion are two-color labeled and then hybridized with Arraystar m6A Single Nucleotide Arrays, thus giving the quantitative value on the m6A level at single nucleotide resolution (Fig. 2). This method does not use semi-quantitative m6A-antibody immunoprecipitation and is quantitatively much more accurate.
Figure 1. MazF enzyme cuts at unmethylated (ACA) sequence but not methylated (m6ACA).
Arraystar m6A Single Nucleotide Array experimental procedures include:
• Fragmentation of total RNA and DNA contamination removal by DNase;
• MazF digestion to cleave unmethylated (ACA) sites while leaving methylated (m6ACA) intact. An aliquot of undigested RNA is used as the Input;
• Reference RNA spike-ins added for hybridization signal normalization;
• cRNA synthesis from the RNA, with Cy5 (red) labeling for the digested and Cy3 (green) for the undigested RNA;
• Hybridization of Cy5- and Cy3-cRNA mixture to Arraystar m6A Single Nucleotide Array.
• Two-color channel microarray data analysis and annotation.
Figure 2. The workflow and procedures of Arraystar m6A Single Nucleotide Array.
Human m6A Single Nucleotide Array
| Total number of distinct probes |
14,321 |
| Probe length |
60 nt |
| Single ACA site |
11,237 |
| Multiple ACA sites |
3,084 |
| Cluster ACA sites |
693 |
| M6ACA site sources |
miCLIP dataset (Linder et al., 2015; Ke et al., 2015; Xu et al., 2017; Chen et al., 2015); RMbase database (Schwartz et al., 2013) |
| Array Format |
8 × 15K |
Mouse m6A Single Nucleotide Array
| Total number of distinct probes |
14,319 |
| Probe length |
60nt |
| Single ACA site |
11,120 |
| Multiple ACA sites |
3,199 |
| Cluster ACA sites |
279 |
| M6ACA site sources |
miCLIP dataset (Linder et al., 2015; Ke et al., 2015; Xu et al., 2017; Chen et al., 2015); RMbase database (Schwartz et al., 2013) |
| Array Format |
8 × 15K |
Rat m6A Single Nucleotide Array
| Total number of distinct probes |
14,581 |
| Probe length |
60nt |
| Single ACA site |
11,499 |
| Multiple ACA sites |
3,082 |
| Cluster ACA sites |
2,614 |
| M6ACA site sources |
Mouse M6ACA site homologs (Linder et al., 2015; Ke et al., 2015; Xu et al., 2017; Chen et al., 2015); RMbase database (Schwartz et al., 2013) |
| Array Format |
8 × 15K |
Rich and detailed bioinformatic analyses and annotations are included with the delivered m6A Single Nucleotide Array data, such as m6A modification stoichiometry, m6A abundance level, m6A location at single nucleotide resolution, transcript model regions. They are essential for understanding m6A dynamics, biological functions, molecular mechanisms and disease associations, but not available by other techniques.
Differential m6A-Methylation
Both differential m6A site methylation stoichiometry and differential m6A site abundance between comparison conditions/groups are provided.
Systematic annotation for m6A sites
ProbeID: Probe ID.
TransID: Database accession of the transcript being m6A modified.
Trans_biotype: Transcript biotype: "ncRNA" for noncoding RNA or "protein_coding" for mRNA.
Gene_Symbol: Official gene symbol for the transcript host gene.
m6A_transcript_location: m6A location in the transcript.
m6A_site_Locus: Genomic coordinates of the m6A site.
m6A_location: Transcript model region where the m6A site is located: 5'UTR, CDS or 3'UTR.
m6A_conservation: If the m6A site has conserved orthologous m6A site between human and mouse species.
Transcript_Length: Full length of the transcript.
Transcript_Sequence: Sequence of the transcript, where, Orange = m6A motif; Orange and underlined = m6A position; Uppercase = probe sequence;
m6ACA sites can have single-, multiple- and clustered site configuration patterns in the transcripts. In addition to single-m6ACA sites, multiple or clustered m6A sites are profiled jointly at high resolution and systematically annotated (Additional fee applies).
m6A_site_Locus: List of genomic coordinates of the constituent sites in the multiple-m6ACA site, separated by a semicolon.
m6A_location: Transcript model region where the multippe-m6A site is located: 5'UTR, CDS or 3'UTR.
m6A_conservation: If the multiple-m6ACA site has conserved orthologous site between human and mouse species.
Transcript_Length: Full length of the transcript.
Transcript_Sequence: Sequence of the transcript, where, Orange = m6A motif, Underlined = m6A position, Uppercase = probe sequence.
Cluster_left: Left boundary of the m6A cluster in the transcript.
Cluster_right: Right boundary of the m6A cluster in the transcript.
Cluster_length: Length of the m6A cluster.
Transcript_sequence: Sequence of the transcript, where,
Orange = MazF cleavable m6A motif, for probe detection;
Pink = MazF uncleavable m6A motif, for defining m6A cluster only;
Underlined = m6A position;
Uppercase = Cluster sequence, which starts and ends with m6A positions in the first and the last m6A motifs.
m6A_site_Locus: List of genomic coordinates of the m6A sites, separated by a semicolon.
m6A_location: List of transcript model region where each constituent m6ACA site resides ( 5'UTR, CDS or 3'UTR), separated by a semicolon.
m6A_conservation: If the m6A site has conserved orthologous m6A site between human and mouse species.
Specific RNA m6A modification sites in bone marrow mesenchymal stem cells from the jawbone marrow of type 2 diabetes patients with dental implant failure. Yan W,et al. International Journal of Oral Science, 2023