新品：RNA m6A甲基化定量试剂盒（比色法）-Epigentek

 EpiQuikTM RNA m6A甲基化定量试剂盒（比色法）(EpiQuik™ m6A RNA Methylation Quantification Kit (Colorimetric)

Epigentek货号：P-9005-48（48个反应），P-9005-96（96个反应），启维益成货号：E159105

本品用比色法定量检测RNA的N6-甲基腺嘌呤（m6A）。适用于任何物种来源如哺乳动物、植物、真菌、细菌和病毒的总RNA。

使用本品的实验仅需3小时45分；高灵敏度，可检测低至10pg的m6A；>70个核苷酸的RNA可结合在板上，因而可定量检测mRNA和ncRNA（如tRNA, rRNA和snRNA）的m6A；在建议的RNA浓度范围内，优化的抗体和增强液使m6A特异性极高，与非甲基腺嘌呤不发生交叉反应；使用简便，结果可靠，分析条件一致。

N6-甲基腺嘌呤（m6A）是真核生物RNA分子最常见和最多的修饰。m6A甲基化修饰由甲基转移酶复合物METTL3催化，去甲基化由最近发现的m6A RNA去甲基酶FTO和ALKBH5催化，去甲基化需要a-酮戊二酸（a-KG）-和二价铁离子（Fe2+）。研究显示METTL3、FTO和ALKBH5在很多生物过程中起重要作用，从发育和代谢到繁殖。80%的RNA甲基化是m6A，m6A存在于多个物种中。它主要分布于mRNA，也存在于非编码RNA如tRNA、rRNA和snRNA。mRNA转录物中m6A的相对丰度影响RNA代谢过程，如剪接、核外运、翻译能力和稳定性，及RNA转录。m6A RNA甲基酶和去甲基酶缺陷引起的异常m6A甲基化水平，导致RNA功能异常及疾病。例如，携带FTO变异而使FTO活性提高的病人体内，异常低水平的靶mRNA m6A与肥胖和糖尿病的发病有关，其通路尚未明确。RNA m6A动态和可逆的化学修饰也可成为具有极大生物学重要性的表观遗传标记物。因此更好的研究m6A RNA甲基化水平和其在RNA转录物上的分布有助于疾病诊断和治疗。

Product Overview
The EpiQuik™ m6A RNA Methylation Quantification Kit (Colorimetric) is a complete set of optimized buffers ａnd reagents to colorimetrically quantify N6-methyladenosine (m6A) in RNA. It is suitable for a direct detection of m6A RNA methylation status using total RNA isolated from any species such as mammals, plants, fungi, bacteria, ａnd viruses. The kit has the following advantages ａnd features:
Colorimetric ELISA-like assay using an m6A antibody with easy-to-follow steps for convenience ａnd speed. The entire procedure can be completed within 3 hours ａnd 45 minutes.
High sensitivity, of which the detection limit can be as low as 10 pg of m6A.
Unique binding solution allows RNA >70 nts to be tightly bound to the wells, which enables quantification of m6A from both mRNA ａnd ncRNA such as tRNA, rRNA, ａnd snRNA.
Optimized antibody ａnd enhancer solutions allow high specificity to m6A, with no cross-reactivity to unmethylated adenosine within the indicated concentration range of the sample RNA.
Universal positive ａnd negative controls are included, which are suitable for quantifying m6A from any species.
Strip-well microplate format makes the assay flexible for manual or high throughput analysis.
Simple, reliable, ａnd consistent assay conditions.

Background Information
N6-methyladenosine (m6A) is the most common ａnd abundant modification in RNA molecules present in eukaryotes. The m6A modification is catalyzed by a methyltransferase complex METTL3 ａnd removed by the recently discovered m6A RNA demethylases FTO ａnd ALKBH5, which catalyze m6A demethylation in an α-ketoglutarate (α-KG)- ａnd Fe2+-dependent manner. It was shown that METTL3, FTO, ａnd ALKBH5 play important roles in many biological processes, ranging from development ａnd metabolism to fertility. m6A accounts for more than 80% of all RNA base methylations ａnd exists in various species. m6A is mainly distributed in mRNA ａnd also occurs in non-coding RNA such as tRNA, rRNA, ａnd snRNA. The relative abundance of m6A in mRNA transcripts has been shown to affect RNA metabolism processes such as splicing, nuclear export, translation ability ａnd stability, ａnd RNA transcription. Abnormal m6A methylation levels induced by defects in m6A RNA methylase ａnd demethylase could lead to dysfunction of RNA ａnd diseases. For example, abnormally low levels of m6A in target mRNAs due to increased FTO activity in patients with FTO mutations, through an as-yet undefined pathway, contributes to the onset of obesity ａnd related diseases. The dynamic ａnd reversible chemical m6A modification in RNA may also serve as a novel epigenetic marker of profound biological significance. Therefore, more useful information for a better understanding of m6A RNA methylation levels ａnd distribution on RNA transcripts could benefit diagnostics ａnd therapeutics of disease.

Principle & Procedure
In this assay, total RNA is bound to strip wells using a RNA high binding solution. N6-methyladenosine (m6A) is detected using a specific capture N6-methyladenosine antibody ａnd detection antibody. The detected signal is enhanced ａnd then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer at a wavelength of 450 nm. The amount of m6A is proportional to the OD intensity measured. Both negative ａnd positive RNA controls are provided in this kit. A standard curve can be performed (range: 0.02 to 1 ng of m6A) or a single quantity of m6A can be used as a positive control. Because m6A content can vary from tissue to tissue, ａnd from normal ａnd diseased states, or vary under treated ａnd untreated conditions, it is advised to run replicate samples to ensure that the signal generated is validated. This kit will allow the user to quantify an absolute amount of m6A ａnd determine the relative m6A RNA methylation states of two different RNA samples.

Starting Materials
Starting materials can include various tissue or cell samples such as cells from flask or microplate cultured cells, fresh ａnd frozen tissues, paraffin-embedded tissues, blood, body fluid samples, etc.

                               
Fig. 1. Schematic procedure of the EpiQuik™ m6A RNA Methylation Quantification Kit (Colorimetric)

                                 
Fig. 2. m6A standard control was added into the assay wells at different concentrations ａnd then measured with the EpiQuik™ m6A RNA Methylation Quantification Kit (Colorimetric).

                             
Fig. 3. Quantification of m6A RNA methylation in different samples. 200 ng of RNA isolated from different tissues or cells were added into the assay wells ａnd the m6A contained in RNA was measured using the EpiQuik™ m6A RNA Methylation Quantification Kit