m6A RNA甲基化片段富集试剂盒(qPCR版)【MeRIP试剂盒】---非常适合qPCR检测单个位点 货号：A-P-9018
EpiQuik CUT&RUN m6A RNA Enrichment Kit
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背景资料：N6-methyl-adenosine (m6A) is the most common and abundant modification on RNA molecules present in eukaryotes. The m6A modification is catalyzed by a methyltransferase complex METTL3 and removed by m6A RNA demethylases FTO and ALKBH5, which catalyze m6A demethylation in an α-ketoglutarate (α-KG)- and Fe2+-dependent manner. METTL3, FTO, and ALKBH5 are known to 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 cause disease. 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 on 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。
产品描述：Kit contains all necessary reagents required for carrying out a successful m6A RNA enrichment starting from total RNA. In the reaction, RNA sequences in both ends of the target m6A-containing regions are cleaved/removed and the target m6A-containing fragments are pulled down using a beads-bound m6A capture antibody. The enriched RNA is then released, purified and eluted. Included in the kit are a non-immune IgG control and m6A positive control. These can be used to demonstrate the efficacy of the kit and performance at the enriched RNA quantification or bioanalyzer step。
产品特点：
1·High enrichment: Uses an RNA cleavage enzyme mix to simultaneously fragment RNA and cleave/remove any RNA sequences in both ends of the target m6A-containing sequences without affecting RNA regions occupied by the antibody. Short RNA fragments are generated only bound with anti- m6A antibody. True target m6A-enriched regions can, therefore, be reliably identified, and high-resolution mapping achieved；
2·Low input: Unbound RNA cleavage and immunocapture are processed in the same single-tube, which enables maximum protection of the target m6A-containing regions and minimized sample loss, allowing the input RNA to be as low as 500 ng；
3.Minimal Background: Cleavage of unbound RNA sequences in the two (2) ends of the target m6A-containing sequences enables minimized MeRIP/sequencing background, allowing data analysis with <10 million reads.
4.Fast, streamlined procedure: The procedure from RNA to library cDNA is less than 3 hours with <30 min of hands-on time；
5.Highly convenient: The kit contains all required components for each step of the EpiQuik™ CUT&RUN m6A RNA Enrichment Kit, which are sufficient for m6A-containing RNA sequence capture, thereby allowing CUTARUNTM m6A RNA enrichment to be the most convenient with reliable and consistent results。
数据分析：
| 操作流程 |
柱状图分析 |
片段分析 |
 |
 |
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| m6A RNA甲基化片段富集试剂盒步骤 |
使用m6A RNA甲基化片段富集试剂盒富集m6A RNA,通过采用A-1801-m6A抗体从10ug人类总RNA和500ng 阳性对照oligo中富集包含m6A 片段的RNA.Non-immune IgG作为阴性对照.对富集后的RNA进行纯化和荧光定量，进行富集倍数比较. |
文库片段分布:通过采用A-1801-m6A抗体从5ug人类总RNA中富集m6A 片段,并进行cDNA文库制备,峰值为195bps，反映了m6A抗体结合RNA的插入大小. |
参考文献：
Ji et. al. (April 2022). ALKBH5-induced circRNA NRIP1 promotes glycolysis in thyroid cancer cells by targeting the miR-541-5p/PKM2 and miR-3064-5p/PKM2 axes Research Square.
Huang J et. al. (January 2022). FTO suppresses glycolysis and growth of papillary thyroid cancer via decreasing stability of APOE mRNA in an N6-methyladenosine-dependent manner. J Exp Clin Cancer Res. 41(1):42.
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。
常见问题：
1.Q: 如何判定富集下来m6A的效率呢?
A:如果要判断m6A富集效率，厂家建议做荧光定量分析，而不是做PCR,因为富集下来的其实也是RNA。
2.Q:富集下来的m6A 片段大概在多少呢?引物设计大概在多么大片段合适呢?
A:如果做qPCR，其实也是做RT-qPCR，先要逆转录为cDNA，富集下来的RNA片段长度为50-200nts，建议引物的扩增长度为70-150bps，qPCR的Ct值取决于RNA的富集程度以及逆转录效果，一般来说，用这个试剂盒来富集5ug小鼠脑RNA样本的话Ct值为30-32，阴性对照的Ct值＞35。
3.Q:能否提供阳性对照的序列?
A:阳性对照的Positive control oligo因为版权原因，是保密的，暂不予提供。
定购信息：
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