Description
Principle of the Assay: The m6A ELISA kit is a competitive ELISA for the quantitative measurement of m6A. The unknown m6A samples or m6A standards are first added to an m6A conjugate preabsorbed microplate. After a brief incubation, an anti-m6A monoclonal antibody is added, followed by an HRP conjugated secondary antibody. The m6A content in unknown samples is determined by comparison with a predetermined m6A standard curve:Background/Introduction: N6-Methyladenosine (m6A) is a nucleoside modification in RNA (and in DNA at extremely low levels) found in most eukaryotes as well as some viruses. m6A is also found in tRNA, rRNA, small nuclear RNA (snRNA) as well as several long non-coding RNA (ncRNA). A large m6A methyltransferase complex directs methylation of adenosine. The complex includes METTL3 which binds s-adenosylmethionine (SAM). Additional components of methyltransferase complex in mammals include METTL14, Wilms tumor 1 associated protein (WTAP) and KIAA1429. More recently, fat mass and obesity-associated protein (FTO) as well as demethylase alkB homolog 5 (ALKBH5) have been identified as m6A demethylases. Links between m6A and numerous cancer types have been identified such as stomach prostate, breast, pancreatic, and kidney cancer, as well as mesothelioma, sarcoma, and leukemia. Reduction in METTL3 levels has been shown to cause apoptosis of cancer cells and reduce invasiveness of cancer cells, while increased ALKBH5 activity by hypoxia was shown to cause increase cancer stem cell numbers. m6A has also been implicated energy homeostasis and obesity regulation since FTO is a key regulator of energy metabolism and obesity. Furthermore, neurodegenerative diseases may be influenced by m6A since dopamine signaling was shown to be FTO dependent. ZC3H13, yet another member of the m6A methyltransferase complex, greatly inhibited growth of colorectal cancer cells when knocked down. Additionally, m6A affects viral infectivity. RNA viruses such as SV40, adenovirus, herpes virus, Rous sarcoma virus, and influenza virus contain m6A methylation on virus genomic RNA. Regulators of m6A control the efficiency of infection and replication of RNA viruses like human immunodeficiency virus (HIV), hepatitis C virus (HCV), and Zika virus (ZIKV). m6A and its cognate factors play crucial roles in regulating virus life cycle and host-viral interactions. The m6A ELISA Kit is a competitive enzyme immunoassay developed for rapid detection and quantitation of m6A in urine, serum, cell or tissue samples. The quantity of m6A in unknown samples is determined by comparing its absorbance with that of a known m6A standard curve. The kit has detection sensitivity limit of 80 ng/mL m6A. Each m6A ELISA Kit provides sufficient reagents to perform up to 96 assays, including standard curve and unknown samples