总体RNA甲基化极易定量检测试剂盒（荧光法） 货号：A-P-9009
MethylFlash 5-mC RNA Methylation ELISA Easy Kit (Fluorometric)
背景资料：5-methylcytosine (5-mC) in DNA occurs by the covalent addition of a methyl group at the 5-carbon of the cytosine ring by DNA methyltransferases. This process has been well studied and is generally associated with repression of gene expression. It was also observed that in humans, 5-mC occurs in various RNA molecules including tRNAs, rRNAs, mRNAs and non-coding RNAs (ncRNAs). 5-mC seems to be enriched in some classes of ncRNA, but relatively depleted in mRNAs. Levels of 5-mC are variable in animal genomes, ranging from undetectable amounts in some insects to about 0.1-0.45% of total RNA in human cells. The majority (83%) of 5-mC sites were found in mRNAs. Within these transcripts 5-mC appears to be depleted within protein coding sequences but enriched in 5’ and 3’ UTRs. Two different methyltransferases, NSUN2 and Dnmt2 are known to catalyze 5-mC modification in eukaryotic RNA. There has been strong evidence that RNA cytosine methylation affects the regulation of various biological processes such as RNA stability and mRNA translation. Furthermore, loss of 5-mC in vault RNAs causes aberrant processing into Argonaute-associated small RNA fragments that can function as microRNAs. Thus, impaired processing of vault ncRNA may contribute to the etiology of human disorders related to NSun2-defciency。
产品描述：This kit contains all reagents necessary for the quantification of global 5-mC RNA methylation. In the assay, RNA is bound to strip-wells that are specifically treated to have a high nucleic acid affinity. 5-mC in RNA is detected using capture and detection antibodies and then quantified fluorometrically by reading the fluorescence in a microplate spectrophotometer. The percentage of 5-mC RNA is proportional to the fluorescence intensity measured。
产品特点：
1·Fast - The entire procedure only needs 2 hours and 40 minutes；
2·Robust - The kit composition allows the assay to have a large “signal window” with less variation between replicates ；
3.Convenient - Inherently low background noise, thereby eliminating the need for plate blocking steps；
4.Sensitive - Detection limit can be as low as 0.02% of 5-mC RNA from 200 ng of input RNA；
5.Specific - High specificity to 5-mC, with no cross-reactivity to unmethylated cytosine or hydroxymethylated cytosine within the indicated concentration range of the sample RNA；
6.Universal - Positive and negative controls allow detection of 5-mC RNA methylation in any species；
7.Accurate - Optimized positive controls that can be fractionalized in percentage scale, allowing the assay to be more accurate and highly comparable with HPLC-MS analysis；
8.Flexible - Strip-well microplate format makes the assay available for manual or high throughput analysis。
数据分析：
| 操作流程 |
标准曲线 |
柱状图分析 |
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| 总体RNA甲基化极易定量检测试剂盒（荧光法）步骤 |
使用5-mC阳性对照绘制标准曲线 |
使用总体RNA甲基化极易定量检测试剂盒（荧光法）精确定量来自各种种属的RNA。结果与MS-LC一致。甚至更好。 |
参考文献：
1.Lewinska A et. al. (August 2017). Reduced levels of methyltransferase DNMT2 sensitize human fibroblasts to oxidative stress and DNA damage that is accompanied by changes in proliferation-related miRNA expression. Redox Biol. 14:20-34.
2.Lewinska A et. al. (February 2017). Downregulation of Methyltransferase Dnmt2 Results in Condition-dependent Telomere Shortening and Senescence or Apoptosis in Mouse Fibroblasts. J Cell Physiol.
保存建议：在接收到艾德寄出的试剂盒后请按照说明书建议，使用不同的保存条件或温度来保存试剂盒内组分。
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表观遗传学产品全面解决方案列下：
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关于5-hmC
5-hmC是近年来在动物组织中发现的，由胞嘧啶修饰而来。5-hmC在表观遗传学上的功能可能与5-甲基化胞嘧啶(5-mC)不同。尽管到现在为止还不确知其功能，有研究者猜测它在调控基因的表达与关闭过程中起着重要的作用。5-mC的发现让我们不得不重新评估DNA甲基信息，也不得不监测人类的健康组织和病理组织之间5-mC相对分布的差异。在EPI公司的MethylFlash技术之前，我们还没有发现任何直接的常规方法来检测5- hmC,以及区分5-hmC和5-mC
5-hmC 和 5-mC的区别
时下常用的DNA甲基化分析方法包括限制内切酶酶切和亚硫酸氢盐或MeDIP介导的MS-PCR和测序，这些技术都不适合用来检测5-hmC，因为它与5-mC事实上很难用这类方法区分开来。为了解决这个问题，EPIk研制了MethylFlash羟甲基化DNA定量试剂盒(荧光法)。本试剂盒提供了一种很经济的方法来检测5-羟甲基化胞嘧啶，并且区分5-hmC, 5-mC, 和 C，使得研究者能够重新评估他们的DNA甲基化信息，也能够在新样品中寻找DNA羟甲基化。 |
