m6A RNA甲基化片段富集试剂盒(qPCR版)【MeRIP试剂盒】
m6A RNA甲基化片段富集试剂盒(qPCR版)，MeRIP试剂盒，MeRIP-qPCR试剂盒，MeRIP Kit，EpiQuik CUT&RUN m6A RNA Enrichment (MeRIP) Kit，基于CUT&RUN技术开发，非常适合从广泛的物种如哺乳类动物，植物，真菌和细菌，还包括培养细胞与新鲜和冷冻的组织等感兴趣的样本中提取总RNA来富集片段化的含有m6A的RNA。少至500ng的总RNA也可开展。整个实验富集时间不到3小时。手动操作不到30分钟。 更多视频请关注视频号【艾维缔】。哔哩哔哩【IVDSHOW】。抖音【军哥聊表观】。
关键词
m6A RNA甲基化片段富集试剂盒(qPCR版)
MeRIP试剂盒
MeRIP-qPCR试剂盒
目录号
A-P-9018
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众所周知，N6-甲基腺苷(m6A)是真核生物RNA分子中最常见和最丰富的修饰。由甲基转移酶复合物METTL3催化m6A修饰，并通过M6A RNA去甲基化酶FTO和ALKBH5，以α-酮戊二酸(α-KG)-和Fe2+-依赖的方式催化M6A去甲基化。METTL3、FTO和ALKBH5在许多生物中起着重要作用，从发育和代谢到生育的过程。在所有RNA碱基甲基化中，m6A占80%以上，存在于各种物种中。m6A主要分布在mRNA中同样也发生在非编码RNA中，如tRNA、rRNA和snRNA。在mRNA转录物中m6A的相对丰度已被证明影响RNA代谢过程，如剪接，核输出，翻译能力和稳定性，以及RNA转录。由m6A RNA甲基酶和脱甲基酶缺陷引起的m6A甲基化水平异常可能导致RNA功能障碍并引起疾病。例如，由于FTO突变患者的FTO活性增加，靶m RNA中m6A的异常低水平，通过一种未定义的途径，导致肥胖和相关疾病的发生。对RNA的化学M6A修饰的动态性和可逆性，也可作为一种具有深远生物学意义的新表观遗传标记。因此，更多有用的信息，以更好地理解m6A RNA甲基氧化RNA 转录物的水平和分布有利于疾病的诊断和治疗。目前，有几种方法被用于表位范围的m6A映射。这些方法包括MeRIP，PA-m6A-seq，miCLIP和m6A-CLIP。MeRIP已被广泛应用，但在m6A分析中无法实现高分辨率。PA-m6A-seq、mi CLIP和m6A-CLIP提高了分析分辨率，但由于重复性差和工艺复杂。特别是，这些方法耗时（>2天）和费用昂贵。为了解决这些问题，IVD Technology Corporation联合开发了一种新的方法：CUT&RUN M6A富集【 (利用核酸酶对m6A进行裂解和回收)】。我们的创新方法将 MeRIP 和 m6A-CLIP 的优点与最快的程序结合起来特别的推出了:m6A RNA甲基化片段富集试剂盒（qPCR版）【简称：MeRIP试剂盒】。
m6A RNA甲基化片段富集试剂盒（qPCR版）【简称：MeRIP试剂盒】 具有以下优势和特点：- 高度富集：使用RNA 切割酶混合物同时对含有m6A 的目标序列两端的RNA 进行片段化和切割/移除任何RNA 序列，而不影响被抗体占据的RNA 区域。短片段RNA 仅与抗m6A 抗体结合。因此，非常可靠地确定真正的m6A 富集目标区域，并可实现高分辨率绘图。
- 低输入：不结合的RNA 切割和免疫捕获是在同一个单管中处理的，这样可最大限度地保护含有m6A的目标区域，并最小化样本损失，允许输入RNA 低至500ng。
- 极小背景：在含m6A 目标序列的两(2)端切割非结合RNA 序列，可最小化MeRIP/测序背景，允许1000万读的数据分析。
- 超快速、精简程序： 从RNA 到cDNA 文库的过程小于3 小时，动手时间<30 分钟。
- 超级方便： 本试剂盒包含了每一步所需的所有组件，这足以捕获含m6A 的RNA 序列，从而允许以一种很方便的方法来富集，结果可靠一致。
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产品组分
*在开盖使用之前，为了最大程度的利用产品，需将量少溶液离心至管底。组分内容
型号：A-P-9018-24(24次)
型号：A-P-9018-48（48次）
型号：A-P-9018-96（96次） 保存条件
WB (Wash Buffer)
30ml
60ml
120ml
4°C
ICB (Immuno Capture Buffer)
5ml
10ml
20ml
常温
NDE (Nuclear Digestion Enhancer)
300ul
600ul
1200ul
常温
CEM (Cleavage Enzyme Mix)*
60ul
120ul
240ul
–20°C
m6A Antibody (1mg/ml)*
50 ul
100ul
200ul
–20°C
Non-Immune IgG (1mg/ml) *
20ul
40ul
80ul
4°C
m6A-Positive Control (200ug/ml) *
6ul
12ul
24ul
–20°C
PDB (Protein Digestion Buffer)
5 ml
10 ml
20 ml
常温
Proteinase K (10mg/ml) *
100ul
200ul
400ul
4°C
Affinity Beads*
100ul
200ul
400ul
4°C
RPS (RNA Purification Solution)
600ul
1.2ml
2.4ml
4°C
RNA Binding Beads *
60ul
120ul
240ul
4°C
Elution Buffer
2000ul
4000ul
8000ul
常温 使用手册
1
1
1 常温
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文件资源
文件下载 文件内容 资源说明 产品视频 A-P-9018-m6A RNA甲基化片段富集试剂盒（qPCR版)【简称：MeRIP试剂盒】 操作手册 有关MeRIP-qPCR引物设计 有关MeRIP-qPCR相对表达量计算 宣传单页 -
营销中心
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注意事项
保存建议【配套现货产品推荐如下】 推荐蓝冰或冰袋运输。当您收到产品后，请严格按照说明书建议保存各产品组分。 如提纯的总RNA是采用trizol溶液的方法 推荐：RTSHOW gDNA去除试剂盒【可从提纯的总RNA中有效的去除gDNA，改善qPCR实验数据】 miRNA纯化试剂盒 推荐：miRNA纯化试剂盒(R-5007)【可从细胞或组织样本中分离20-200nt的miRNA，siRNA, snRNA等小片段RNA】 mRNA纯化试剂盒 推荐：mRNA纯化试剂盒(Z5300)【针对纯化的总RNA来进行】IVDSHOW Oligo(dT)18磁珠(R-9100)【针对细胞和组织样本】 磁珠法纯化 推荐：病毒DNA/RNA提取试剂盒(磁珠法）(SBK186)【针对血清，血浆，鼻拭子，鼻咽拭子，唾液等体液样本来纯化】 RNA纯化试剂盒 推荐：总RNA纯化试剂盒(TR01)【据不同的样本来选型，比如纯化病毒RNA选型指南】 植物样本RNA纯化试剂盒 推荐：植物总RNA纯化试剂盒(TR02)【采用离心柱法纯化RNA选型指南】 cDNA反转录试剂盒【pg~ng级别】 推荐：IVDSHOW 高保真 RT Plus Master Mix(R-G207)【针对富集的片段化m6A进行反转录成cDNA】 cDNA反转录试剂盒 推荐：IVDSHOW 高保真 RT All-in-One Master Mix(R-G208)【针对富集的片段化m6A进行反转录成cDNA】 qPCR试剂盒 推荐：EvaGreen qPCR PreMix(R-EMX)【据实时荧光定量PCR仪来选型】 IVDSHOW 16孔合金磁力架 推荐：IVDSHOW 16孔合金磁力架(C3111)【可放置16x0.2ml PCR管，非常适合小体积洗脱，少至5ul也可操作】 IVDSHOW 16孔磁力架 推荐：IVDSHOW 16孔磁力架(C3001)【可放置16x0.2ml PCR管】快速MS-qPCR试剂盒【甲基化专用酶】 IVDSHOW 96孔磁力架 推荐：IVDSHOW 96孔磁力架(C3004)【可放置96孔 PCR板，性价比超高】 m6A兔源多克隆抗体【MeRIP级】 推荐：m6A兔源多克隆抗体【MeRIP级】(A-1801)【可做DB,ELISA,IF,IP和MeRIP】 m6A兔源多克隆抗体[2H6]【MeRIP级】 推荐：m6A兔源单克隆抗体【MeRIP级】(A-1802)【可做DB,ELISA,IF,IP和MeRIP】 -
FAQ
扩展阅读：CUT&RUN技术是如何帮助提高m6A RNA甲基化免疫沉淀的效果?
Q:N6-methyladenosine (m6A or 6mA)分子式是什么?
A:
这个就是N6-methyladenosine (m6A)分子式，也就是N6-甲基腺嘌呤，简称：m6A or 6mA。我们也单独出售这种化合物【More】Q:CUT&RUN是什么呢?
A:CUT&RUN【Cleavage under targets and release using nuclease】，CUT&RUN具有显著优势，该技术实验周期短，信噪比高，可重复性好，且细胞投入量低，尤其适用于早期胚胎发育、干细胞、肿瘤以及表观遗传学等研究领域。【More】
Q:m6A RNA【RNA中含有的m6A】 是否等同于 6mA or m6A DNA【DNA中含有的6mA or m6A】？
A:m6A RNA和6mA DNA取决于许多因素，包括物种、正常与疾病状态、组织类型(如大脑、肝脏)等。您所掌握的搜索文献可能有助于获得有关其特定样品的更多信息。
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，我们建议做荧光定量分析，而不是做PCR，因为富集下来的也是RNA。
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，即RT-qPCR，首先将富集下来的m6A片段逆转录为cDNA，富集下来的m6A RNA片段长度约为50-200nts，建议qPCR引物的设计覆盖70-150bps扩增长度，且包含预期有m6A修饰的区域。通常可借助引物设计软件如：Primer3。qPCR的Ct值取决于m6A 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【Non-immune IgG】的Ct值＞35。
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。
Q:试剂盒提供的阳性对照能做几次？同步实验后的富集百分比大概在多少呢？
A:本试剂盒同步实验够2次使用，每次3ul用量；根据我们的质量控制测试，通常每次阳性对照【200ng/ul,每次3ul用量】的富集效率约为总输入阳性对照【200ng/ul,每次3ul用量】的4-7％。如本试剂盒中每次阳性对照需加3ul，如果按照4-7%计算所得阳性对照富集量大概是：24-42ng/ul这个范围。
Q:关于操作手册中1d-1e中间划线部分的说明？
A:这一步是用来做目的基因IP后qPCR对照的，做qPCR的对照来使用，相当于起内参基因的作用。没有与抗体一起孵育，不用做1a-d，直接酶解。然后进入2d，这个input组加的RPS为25ul。
Q:关于组分m6A-Positive Control 说明？
A:它不包含引物，是一种合成的寡核苷酸，没有种属特异性。主要用于评估试剂盒本身的富集效率的。
Q:在提纯总RNA过程中，是否需要消化DNA？
A:为了尽可能减少DNA中m6A的干扰得到较好的实验结果，我们建议您纯化总RNA时，尽可能采用DNase I【类似的有去除DNA功能的试剂盒如：SpinSHOW 柱上消化试剂盒】对于DNA进行消化处理。或者选购我们针对细胞和组织样本配套的总RNA纯化试剂盒【TR01】其他样本类型可以直接搜索官网查询。
Q:如您采用的是mRNA标本是否可以采用这个盒子呢？
A:本试剂盒的输入样本是提取的RNA，可从任何物种和各种来源(如培养细胞、新鲜/冷冻组织、石蜡包埋组织、血液、体液样本等)中分离出来。纯化的、完整的mRNA也可以作为这个试剂盒的输入材料。RNA应该在无RNase的水或合适的缓冲液(例如，TE)中，并且应该是高质量和相对无DNA的。
Q:如果我采用的是大肠杆菌的样本，是否可以采用这款产品？
A:这款产品我们并非针对此标本专门设计的，暂时还没有验证过。因为这款产品针对的是位点设计的，而非针对物种。一般来说，是可以使用的。
Q:这个富集试剂盒。是只能富集总RNA中的m6A吗？还是说DNA中的m6A也能富集到呢？
A:本试剂盒中提供的m6A抗体可以检测到DNA上的5-mC，因为该抗体对m6A修饰具有特异性。因此，应将纯化的RNA作为起始材料，如本产品指南所述。目前还没有把DNA作为起始材料来富集。
Q:试剂盒中的单独组分：m6A Antibody 是否单独出售？是单克隆抗体，还是多克隆抗体？
A:可以单独定购。型号：A-P-9018-24-m6A 品名：m6A Antibody (1 mg/ml)* 规格：50 µl；试剂盒中的m6A Antibody 是多克隆抗体。也可以选择m6A兔源多克隆抗体【A-1802】和m6A兔源单克隆抗体[2H6]【A-1801】
Q:在MeRIP项目中，为什么Input组和IP组都要做？
MeRIP项目中Input和IP组成一对样品。实验环节IP样品用m6A抗体特异性富集甲基化修饰的RNA片段，而Input仅仅是片段化的RNA则作为对照消减背景噪音，在建库、测序平行开展。结合峰检测分析需整合两个样本的数据，并利用Input数据排除本底表达水平高或非特异性结合的peaks，以提高calling peak的准确性。
【操作层面的注意事项】
Q:试剂盒中的单独组分：ICB是否够用？
A:a).与抗体的反应最多需要189ul ICB（样品+ m6A抗体或非免疫IgG rxns）或191ul ICB（阳性对照寡核苷酸+ m6A抗体rxn）。
b).“Input输入”反应（无抗体对照的rxns）仅需要20ul ICB。
c).因此，对于具有3种条件的8个样品（m6A抗体-189 ul ICB；非免疫IgG-189 ul ICB；“输入”-20 ul ICB），需要24个ICB的总体积 反应（8个样品* 3个条件，每个= 24 rxns）为8 *（189 + 189 + 20）= 3.2 ml，足够了。
d). ICB通常会提供额外的容量（〜0.5 ml）。Q:针对步骤2b中热循环仪是否盖盖子？
A:对于在没有加热盖子的热循环仪中进行的步骤2b孵化，是指关闭盖子的热量。 如果热循环仪没有这个选项，就在没有盖子的情况下或不盖盖子的情况下，进行孵化反应。
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