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m6A RNA甲基化定量检测试剂盒(比色法)
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p-9005-19
p-9005-16

m6A RNA甲基化定量检测试剂盒(比色法)

m6A RNA甲基化定量检测试剂盒(比色法) 专门为定量检测总RNA,mRNA和非编码RNA中m6A RNA甲基化水平而设计的。非常适合对总RNA,mRNA和非编码RNA进行m6A RNA甲基化总体水平定量分析,整个实验时间仅需3.5小时。 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】。
p-9005-19
p-9005-16
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文献追踪

 众所周知,在真核生物中,m6A (N6-methyladenosine)是在RNA分子中最常见的和丰富存在的。这种修饰是由m6A催化甲基转移酶复杂METTL3和最近发的m6A RNA 去甲基转移酶如FTO和ALKBH5,这种 m6A RNA去甲基是以α-ketoglutarate m6A(α-KG)-和Fe2+-方式进行的。 结果表明, 在许多生物过程中,如从生命发展和代谢生育,METTL3,FTO和ALKBH5扮演着重要角色。超过80%的m6A是以RNA 甲基化的形式存在于不同的物种。 m6A主要分布在mRNA和也发生在非编码RNA(ncRNA)如tRNA,rRNA和snRNA。相对丰富的m6A在mRNA转录时已经表明影响核糖核酸代谢过程,如拼接,核出口,翻译能力和稳定性,RNA转录。异常甲基化水平的m6A诱导缺陷甲基化酶和m6A RNA去甲基酶可能导致RNA功能障碍的和疾病发生。例如,异常低水平在正常m6A目标由于增加患者FTO活动、FTO基因突变,通过迄今还未通路,导致了肥胖和相关疾病的发作。

动态和可逆化学m6A修饰,在RNA中也可以作为一种新颖的表观遗传标记的生物学,意义深远可以预见。因此,更多的有用的信息,更好理解m6A RNA甲基化水平和分布对RNA转录、诊断和治疗疾病非常有益。 该产品中,总RNA必将结合在微孔板上,使用一种高特异性的RNA溶液方案。使用特异性较强的捕获抗体和检测抗体来分析m6A。检测产生的增强信号,然后通过读取在微型板分光光度计波长为450 nm处吸光度进行比色定量。m6A的数量与测量的OD值强度是成正比的。在这个试剂盒中包括阳性和阴性对照。绘制标准曲线 (范围:0.02到1 ng的m6A)或单点对照的m6A可以用作阳性对照。因为m6A的含量,在不同的组织,正常和病变的部位,或在处理过以及未处理的条件下,我们建议运行2份样品,以确保信号生成的可信心性。这套解决方案将允许研究人员选择定量一个绝对数量的m6A或确定相对m6A RNA甲基化状态的两个不同的RNA样品。 该款产品拥有一套完整的优化缓冲液和试剂体系,可以采用比色或荧光法来定量总RNA中的 m6A (N6-methyladenosine)。对于总RNA(从哺乳动物,各种组织或细胞样本如像培养瓶和培养皿培养的细胞,新鲜和冷冻的组织,石蜡包埋组织,血液,体液,植物,真菌,细菌和病毒等任何样本中提取的) 中m6A直接定量检测甲基化状态是非常理想的。

在mRNA中m6A甲基化是可逆的(文献1)

对于细胞与组织等样本,m6A RNA甲基化定量检测试剂盒(比色法) 具有以下优势特:

  • 类似于Elisa步骤的比色法检测方便快速。整个操作步骤可在3小时45分钟完成;荧光法更短。
  • 高敏,检测的最低下限为 10 pg 的 m6A;荧光法更低 。
  • 独特的结合液容许>70 nts 的RNA紧紧的结合在孔上,可以定量检测来自mRNA和ncRNA如tRNA,rRNA和snRNA。
  • 优化的抗体和增强剂溶液可以特异的定量检测m6A,对于特定片段腺苷浓度范围的未甲基化样本RNA不会产生交叉反应。
  • 包括的阴性和阳性对照,对于来自任何种属的m6A都可以实现定量检测。
  • 96孔可拆卸板模式使研究人员能根据自己需要选择手工或是高通量模式分析。
  • 操作简便、可信、分析条件始终如一。

产品组分

组分内容

型号:A-P-9005-48(48)

型号:A-P-9005-96(96)

储存温度

WB

10X Wash Buffer

14 ml

28 ml

4°C

BS

Binding Solution

5 ml 10 ml 室温

NC

Negative Control,0% 5-mC,100ug/ml *

10 ul

20 ul

-20°C

PC

Positive Control,m6A,2ug/ml *

10 ul

20 ul

-20°C

CA

Capature Antibody,1000X*

5 ul 10 ul 4°C

DA

Detection Antibody,1000X*

6 ul 12 ul -20°C

ES

Enhancer Solution

5 ul 10 ul -20°C

DS

Developer Solution

5 ml 10 ml 4°C

SS

Stop Solution

5 ml

10 ml

4°C

8-联管

8-Well Assay Strips(With Frame)

6条

12条

4°C

操作手册

 

1

1

室温

*注意:使用各溶液之前,将溶液离心至管底。NC(阴性对照)是一种不含m6A的RNA。PC(阳性对照)是一种含有m6A oligos和含有100% m6A的。

营销中心

注意事项

保存建议 推荐蓝冰或冰袋运输。当您收到产品后,按照说明书建议保存各组分。
警告 本品仅供科研使用,请勿用于临床与诊断。

 

FAQ

Q:什么样的标本可以采用这款产品呢?

A:(1)RNA样本类型:培养的细胞,新鲜和冷冻的组织,石蜡包埋的组织,血液,体验等等样本的总RNA,mRNA,tRNA,rRNA和snRNA均可适用该试剂盒。

(2)RNA样本提取方式:市面上销售的试剂盒提取的均可,Trizol法可能是可以的,但是Trizol法提取的请确保提取后将RNA重新溶解在不含RNase的水中或合适的缓冲液(例如TE)中,并且RNA质量高且相对不含DNA。

(3)RNA样本符合要求的标准:OD 260/280 >1.9。260/230 > 1.7 【无DNA污染】。

Q:试剂盒中提供的标准品如何配置呢?

 A:(1)阳性对照怎么设置

对于第一次上手做这个实验的同学们或许都会有些疑惑,阳性对照难道不都是要做标准曲线的吗?不然怎么对照和计算呢?

其实,这个试剂盒是可以根据实验需要进行选择做单点对照(0.5ng/ml)还是做标准曲线(0.02~1ng/ml)。建议做复孔哟。

(2)阳性对照信号太弱

【1】添加至孔中的PC量不足,确保加入足够量的PC;

【2】标准液未进行最佳稀释,并充分混合;

【3】确保PC的保存方式是按照说明书进行保存的,一定要保存在指定条件下哟,

【4】在使用的时候,要注意稀释后放置在冰上,保证有效成分不被讲解。

(3)阴性对照背景值太高

【1】可能没有洗干净,按照说明书进行清洗;

【2】被PC或者样本污染了,切记要换枪头加样;

【3】孵育时间太长,按照说明书进行操作1~10min,最长也不能超过2h;

【4】显色时间过长,可适当缩减显色时间。

(4)样本值太低或者没信号

【1】可能是因为没有加入足够量的RNA。厂家推荐的样本输入量为100ng~300ng,200ng为最佳,但是这又不是绝对的噢,同学们可根据自己的样本做相应的调整尤其在信号偏弱的情况下,可适当上调RNA的输入量。

【2】可能是因为产品本身m6A的含量就很低。

【3】可能是样本的纯度不够,有DNA或者其他的污染,或者RNA发生了降解,这些都可能造成低信号。

【4】mRNA样本的话,可能因为片段太小(<80 nts),也会导致低信号。

Q:如何进行数据分析呢?

A:(1)如何计算标曲

线性回归,二阶多项式,四参数对数回归都可以。

m6A 如何计算标曲

(2)不同输入量检测出来的数据是否可一同进行数据分析?

是可以的,但是要注意计算的时候要以不同的数据进行。比如你第一次的样本输入量是200ng,计算公式带入的值为200;第二次样本输入量是300ng,计算公式带入的值必须为300.

(3)什么情况下的样本OD值被认为是可用的?

只要样品的OD高于NC的数值,该数据仍可用于计算m6A水平。

(4)重复孔之间差异较大怎么计算?

对于重复孔之间差异较大,我们若是重复孔3个以上的话,我们一般会选择舍弃其中差异特别特别大的一个值后再进行计算,但是只有2个孔的话,该怎么办呢?这个时候我们不能随便舍弃任何一个孔的值,同样也没有办法提供相关的建议。但是需要注意:p-9005比色测定的%CV约为15%。

Q:如何进行稀释?

A:(1)需要稀释的试剂组分如何稀释?

 按照说明书上给的配制比例进行稀释,用TE buffer稀释,建议即用即配,计算好所需的量再进行稀释配制。

(2)为什么有些组分稀释后需要放置在冰上?

使用前应将稀释的成分(例如PC标准品)放在冰上,以最大程度地减少试剂降解并保持试剂完整性。

(3)稀释液必须是TE吗?可以替换成DEPC水或者无RNAase的水吗?

可以使用DEPC水替代TE buffer,无RNAase的水也可以。

Q:其他要点提示?

A:(1)所有组分需要放置在室温条件下回温吗?

   对于产品组份中的Wash buffer,保存在低温下可能会有盐离子等沉淀,在使用前需要将其拿至室温进行回温,沉淀即可溶解,这样可进行正常的实验。

(2)如何减少复孔间差异?

【1】在板中垂直而不是水平地加载复制样品,因为这可以确保同时添加溶液以复制孔,并有助于最小化复制差异。

【2】确保从孔中完全去除洗涤缓冲液,因为任何残留的缓冲液都会增加背景信号和重复变异性,尤其是在敏感的ELISA中(例如用于甲基化检测的ELISA)。

【3】使用手动移液器代替典型的板轻拂/倾倒方法,以确保完全去除缓冲液并最大程度减少孔间污染。

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Wang J et. al. (January 2024). Regulation of ULK1 by WTAP/IGF2BP3 axis enhances mitophagy and progression in epithelial ovarian cancer. Cell Death Dis. 15(1):97.

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Song M et. al. (January 2024). N6 methyladenosine eraser FTO suppresses Staphylococcus aureus-induced ferroptosis of bone marrow mesenchymal stem cells to ameliorate osteomyelitis through regulating the MDM2/TLR4/SLC7A11 signaling pathway. Cell Biol Int.

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Hu C et. al. (December 2023). Putrescine promotes MMP9-induced angiogenesis in skeletal muscle through hydrogen peroxide/METTL3 pathway. Free Radic Biol Med.

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Liu C et. al. (December 2023). ALKBH5 protects against stroke by reducing endoplasmic reticulum stress-dependent inflammation injury via the STAT5/PERK/EIF2α/CHOP signaling pathway in an m(6)A-YTHDF1-dependent manner. Exp Neurol. :114629.

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Huang L et. al. (December 2023). WTAP regulates autophagy in colon cancer cells by inhibiting FLNA through N6-methyladenosine. Cell Adh Migr. 17(1):1-13.

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Wang X et. al. (June 2023). METTL3-mediated m6A modification of SIRT1 mRNA inhibits progression of endometriosis by cellular senescence enhancing. J Transl Med. 21(1):407.

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Yu Z et. al. (June 2023). FTO alleviates cerebral ischemia/reperfusion-induced neuroinflammation by decreasing cGAS mRNA stability in an m6A-dependent manner. Cell Signal. 109:110751.

Liang J et. al. (June 2023). Role of miR-300-3p in Leydig cell function and differentiation: A therapeutic target for obesity-related testosterone deficiency. Mol Ther Nucleic Acids. 32:879-895.

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Zhou Y et. al. (May 2023). Effect of prenatal perfluoroheptanoic acid exposure on spermatogenesis in offspring mice. Ecotoxicol Environ Saf. 260:115072.

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Chen J et. al. (May 2023). METTL3 promotes pancreatic cancer proliferation and stemness by increasing stability of ID2 mRNA in a m6A-dependent manner. Cancer Lett. 565:216222.

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Liu L et. al. (May 2023). m(6)A eraser ALKBH5 mitigates the apoptosis of cardiomyocytes in ischemia reperfusion injury through m(6)A/SIRT1 axis. PeerJ. 11:e15269.

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Chen YH et. al. (May 2023). m(6)A-dependent mevalonate kinase in juvenile hormone synthesis pathway regulates the diapause process of bivoltine silkworm (Bombyx mori). Mol Biol Rep.

Guo Z et. al. (May 2023). Carbon Dots from Lycium barbarum Attenuate Radiation-Induced Bone Injury by Inhibiting Senescence via METTL3/Clip3 in an m(6)A-Dependent Manner. ACS Appl Mater Interfaces. 15(17):20726-20741.

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Fu Y et. al. (April 2023). LncRNA GAS5 regulated by FTO-mediated m6A demethylation promotes autophagic cell death in NSCLC by targeting UPF1/BRD4 axis. Mol Cell Biochem.

An X et. al. (April 2023). ZBTB7C m6A modification incurred by METTL3 aberration promotes osteosarcoma progression. Transl Res.

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Chen X et. al. (April 2023). KIAA1429-mediated m6A modification of CHST11 promotes progression of diffuse large B-cell lymphoma by regulating Hippo-YAP pathway. Cell Mol Biol Lett. 28(1):32.

Zhao Y et. al. (April 2023). METTL16, an evolutionarily conserved m6A methyltransferase member, inhibits the antiviral immune response of miiuy croaker (Miichthys miiuy). Dev Comp Immunol. :104713.

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Kim Y et. al. (April 2023). METTL3 regulates alternative splicing of cell cycle-related genes via crosstalk between mRNA m(6)A modifications and splicing factors. Am J Cancer Res. 13(4):1443-1456.

Fang Q et. al. (April 2023). YTHDF1 phase separation triggers the fate transition of spermatogonial stem cells by activating the IκB-NF-κB-CCND1 axis. Cell Rep. 42(4):112403.

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Xu C et. al. (April 2022). Methyltransferase-Like 3 Rescues the Amyloid-beta protein-Induced Reduction of Activity-Regulated Cytoskeleton Associated Protein Expression <i>via</i> YTHDF1-Dependent N6-Methyladenosine Modification. Front Aging Neurosci. 14:890134.

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Zhu Y et. al. (April 2022). METTL3-mediated m6A modification of STEAP2 mRNA inhibits papillary thyroid cancer progress by blocking the Hedgehog signaling pathway and epithelial-to-mesenchymal transition. Cell Death Dis. 13(4):358.

Wang JN et. al. (April 2022). Inhibition of <i>METTL3</i> attenuates renal injury and inflammation by alleviating <i>TAB3</i> m6A modifications via IGF2BP2-dependent mechanisms. Sci Transl Med. 14(640):eabk2709.

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Liu H et. al. (April 2022). Analysis of the function and mechanism of DIRAS1 in osteosarcoma. Tissue Cell. 76:101794.

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Yang F et. al. (April 2022). Role of N6-methyladenosine RNA modification in the imbalanced inflammatory homeostasis of arsenic-induced skin lesions. Environ Toxicol.

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Wang Y et. al. (March 2022). METTL14 promotes prostate tumorigenesis by inhibiting THBS1 via an m6A-YTHDF2-dependent mechanism. Cell Death Discov. 8(1):143.

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Yang JJ et. al. (March 2022). ALKBH5 ameliorated liver fibrosis and suppressed HSCs activation via triggering PTCH1 activation in an m<sup>6</sup>A dependent manner. Eur J Pharmacol. :174900.

Wu Y et. al. (March 2022). PRMT5 regulates RNA m6A demethylation for doxorubicin sensitivity in breast cancer. Mol Ther.

Yu T et. al. (March 2022). ALKBH5 Promotes Multiple Myeloma Tumorigenicity through inducing m<sup>6</sup>A-demethylation of SAV1 mRNA and Myeloma Stem Cell Phenotype. Int J Biol Sci. 18(6):2235-2248.

Ke WL et. al. (March 2022). m<sup>6</sup>A demethylase FTO regulates the apoptosis and inflammation of cardiomyocytes via YAP1 in ischemia-reperfusion injury. Bioengineered. 13(3):5443-5452.

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Chen J et. al. (February 2022). TBK1-METTL3 axis facilitates antiviral immunity. Cell Rep. 38(7):110373.

Cai Y et. al. (February 2022). METTL3 regulates LPS-induced inflammatory response via the NOD1 signaling pathway. Cell Signal. 93:110283.

Wang et. al. (February 2022). Global N6-Methyladenosine Profiling Revealed the Tissue-Specific Epitranscriptomic Regulation of Rice Responses to Salt Stress Int. J. Mol. Sci.. 23(4)

Chang Y et. al. (February 2022). N<sup>6</sup>-methyladenosine RNA modification of glutamatergic neurons is associated with contextual fear discrimination. Physiol Behav. 248:113741.

Zhao X et. al. (February 2022). N6-methyladenosine-dependent modification of circGARS acts as a new player that promotes SLE progression through the NF-κB/A20 axis. Arthritis Res Ther. 24(1):37.

Hu Y et. al. (February 2022). Demethylase ALKBH5 suppresses invasion of gastric cancer via PKMYT1 m6A modification. Mol Cancer. 21(1):34.

Xie Q et. al. (January 2022). piRNA-14633 promotes cervical cancer cell malignancy in a METTL14-dependent m6A RNA methylation manner. J Transl Med. 20(1):51.

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Xu Y et. al. (January 2022). METTL3 promotes lung adenocarcinoma tumor growth and inhibits ferroptosis by stabilizing SLC7A11 m<sup>6</sup>A modification. Cancer Cell Int. 22(1):11.

Huang Y et. al. (January 2022). Isovitexin protects against acute liver injury by targeting PTEN, PI3K and BiP via modification of m6A. Eur J Pharmacol. 917:174749.

Peng J et. al. (January 2022). The m6A methyltransferase METTL3 affects autophagy and progression of nasopharyngeal carcinoma by regulating the stability of lncRNA ZFAS1. Infect Agent Cancer. 17(1):1.

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Shen D. et. al. (December 2021). METTL14-mediated Lnc-LSG1 m6A modification inhibits clear cell renal cell carcinoma metastasis via regulating ESRP2 ubiquitination Nucleic Acids. 27:547-561.

Wei A et. al. (December 2021). N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) mitigates the liver fibrosis via WTAP/m<sup>6</sup>A/Ptch1 axis through Hedgehog pathway. Gene. 813:146125.

Zhou H et. al. (December 2021). Low Expression of YTH Domain-Containing 1 Promotes Microglial M1 Polarization by Reducing the Stability of Sirtuin 1 mRNA. Front Cell Neurosci. 15:774305.

Li Y et. al. (December 2021). METTL3 facilitates the progression of hepatocellular carcinoma by modulating the m6A level of USP7. Am J Transl Res. 13(12):13423-13437.

Xu A et. al. (December 2021). FTO promotes multiple myeloma progression by posttranscriptional activation of HSF1 in an m<sup>6</sup>A-YTHDF2-dependent manner. Mol Ther.

Zhang X. et. al. (December 2021). N6-methyladenosine modification governs liver glycogenesis by stabilizing the glycogen synthase 2 mRNA Research Square.

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Cheng P et. al. (December 2021). Amelioration of acute myocardial infarction injury through targeted ferritin nanocages loaded with an ALKBH5 inhibitor. Acta Biomater.

Jia et. al. (December 2021). LXA4 Enhances Prostate Cancer Progression Through Facilitating M2 Macrophage Polarization via Inhibition of METTL3 SSRN.

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Ouyang et. al. (November 2021). METTL3 Depletion Contributes to HR+/HER2- Breast Cancer Progression and Drug Resistance via m6A Modification of Constituents of the CDKN1A/EMT and BAX/caspase-9/-3/-8 Signalling Pathways Research Square.

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Chen J et. al. (October 2021). HOTAIR/Sp1/miR-199a critically regulates cancer stemness and malignant progression of cutaneous squamous cell carcinoma. Oncogene.

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Lin S et. al. (September 2021). METTL3-Induced miR-222-3p Upregulation Inhibits STK4 and Promotes the Malignant Behaviors of Thyroid Carcinoma Cells. J Clin Endocrinol Metab.

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Song H et. al. (September 2021). METTL3-mediated m<sup>6</sup>A RNA methylation promotes the anti-tumour immunity of natural killer cells. Nat Commun. 12(1):5522.

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Song Y et. al. (September 2021). METTL3-Mediated lncRNA m<sup>6</sup>A Modification in the Osteogenic Differentiation of Human Adipose-Derived Stem Cells Induced by NEL-Like 1 Protein. Stem Cell Rev Rep.

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Chang M et. al. (September 2021). METTL3-mediated RNA m6A hypermethylation promotes tumorigenesis and GH secretion of pituitary somatotroph adenomas. J Clin Endocrinol Metab.

Ge Y et. al. (September 2021). Degradation of WTAP blocks antiviral responses by reducing the m<sup>6</sup> A levels of IRF3 and IFNAR1 mRNA. EMBO Rep. :e52101.

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