EpiQuik M6A RNA甲基化定量检测试剂盒（比色法）-表观遗传学-其他特色产品-产品中心-上海宇劲生物技术有限公司

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EpiQuik M6A RNA甲基化定量检测试剂盒（比色法）

英文名称：EpiQuik M6A RNA Methylation Quantification Kit(Colorimetric)
中文名称：EpiQuik M6A RNA甲基化定量检测试剂盒（比色法）
供应商：Epigentek
产品货号：P-9005-48/P-9005-96
目录价：5544/9152
规格：48Assays/96Assays

名称：EpiQuik M6A RNA甲基化定量检测试剂盒（比色法）

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·产品描述

N6-甲基腺嘌呤（m6A）是真核生物最常见和最丰富的RNA分子修饰.m6A修饰是METTL3甲基转移酶复合体催化的，而最近新发现的RNA去甲基化酶FTO 和ALKBH5 可以通过一种A-酮戊二酸和Fe2+依赖型的形式对m6A去甲基化.METTL3， FTO和 ALKBH5被证明在很多生物过程中起重要作用，从发育和代谢到生殖.m6A存在于很多物种质中，占到了RNA碱基甲基化修饰的80%.m6A主要分布在mRNA中，也出现在非编码RNA如tRNA， rRNA和snRNA.在转录成的RNA中相对高的m6A含量会影响RNA的代谢过程，比如剪切，核转运，翻译能力和稳定性，以及RNA转录.异常的m6A甲基化程度包括m6A RNA甲基化酶的缺失和去甲基化酶的缺失，这可能导致RNA的失效和疾病.比如，FTO基因突变导致的病人体内很高的FTO活性从而导致低含量的m6A，虽然机制没有完全弄清，但是却导致了肥胖和相关疾病.动态的和可逆的m6A修饰可能是一个起重要作用的新的表观遗传标记.因此，关于m6A更多的有用信息可以使人们对RNA m6A甲基化程度和在转录成的RNA内分布有更好的理解，从而对诊断和治疗疾病有更多的促进。

EpiQuik M6A RNA甲基化定量检测试剂盒（比色法）是一组优化的、完整的的试剂组合，可以通过比色的方法定量RNA中N6-甲基腺嘌呤（m6A）.它可以直接检测哺乳动物，植物，真菌细菌和病毒的总RNA样本的m6A的甲基化程度。

·产品特点

1.ELISA型的检测方法采用了m6A抗体和方便的检测步骤，可以在3小时45分钟内完成检测

2.很高的灵敏度，可以检测到10pg的m6A

3.独特的反应试剂可以是大于70nt的RNA牢固地结合在板子上，从而定量mRNA 和 ncRNA （ tRNA， rRNA， and snRNA）

4.优化的抗体对m6A有很高的特异性，对未甲基化的腺嘌呤没有交叉反应

5.通用的阳性对照和阴性对照适用于任何五中的m6A检测

6.条形的微孔板使得反应可以灵活处理，既可以手动操作，也可以高通量处理

7.简单，可靠和稳定的检测条件

·产品说明书

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 or 6mA) 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
The input material required is RNA, which can be isolated from 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. The amount of RNA for each assay can be 100 ng to 300 ng. For optimal quantification, the input RNA amount should be 200 ng, as the abundance of m6A is generally less than 0.1% of total RNA.

Product Citations

Tsuruta K. et. al. et. al. (September 2020). RNA N6-methyladenosine demethylase FTO regulates PD-L1 expression in colon cancer cells Biochemical and Biophysical Research Communications. 530(1)

Xue L et. al. (September 2020). m6 A transferase METTL3-induced lncRNA ABHD11-AS1 promotes the Warburg effect of non-small-cell lung cancer. J Cell Physiol.

Zhang S et. al. (September 2020). Altered m6 A modification is involved in up-regulated expression of FOXO3 in luteinized granulosa cells of non-obese polycystic ovary syndrome patients. J Cell Mol Med.

Wei J et. al. (August 2020). METTL3 potentiates resistance to cisplatin through m6 A modification of TFAP2C in seminoma. J Cell Mol Med.

Wang M. et al. et. al. (August 2020). Upregulation of METTL14 mediates the elevation of PERP mRNA N 6 adenosine methylation promoting the growth and metastasis of pancreatic cancer Mol Cancer. 19(1)

Luo Q et. al. et. al. (August 2020). Decreased ALKBH5, FTO, and YTHDF2 in Peripheral Blood Are as Risk Factors for Rheumatoid Arthritis Biomed Res Int.

Chen S et. al. (August 2020). WTAP promotes osteosarcoma tumorigenesis by repressing HMBOX1 expression in an m6A-dependent manner. Cell Death Dis. 11(8):659.

Xu Y et. al. (August 2020). The FTO/miR-181b-3p/ARL5B signaling pathway regulates cell migration and invasion in breast cancer. Cancer Commun (Lond).

Du J. et. al. et. al. (August 2020). N 6-Adenosine Methylation of Socs1 mRNA is Required to Sustain the Negative Feedback Control of Macrophage Activation Developmental Cell.

Zhang S et. al. (August 2020). Pregnancy carbon black nanoparticles exposure-induced neurobehavioral deficits are associated with m6A modification in offspring. Neurotoxicology.

Yang Z. et. al. et. al. (August 2020). RNA N6-Methyladenosine Reader IGF2BP3 Regulates Cell Cycle and Angiogenesis in Colon Cancer General Surgery.