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:
• 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>>
• 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 the functional significance of these m6A sites, please click here.
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.
The rich and detailed bioinformatic analyses and annotations delivered by m6A Single Nucleotide Arrays include single nucleotide m6A modification stoichiometry, m6A location, site type (Single-, Multiple-, clustered m6A sites), transcript regions, and so on. They are essential for understanding m6A dynamics, biological functions, molecular mechanisms and disease associations, but not available by other techniques.