EZ RNA甲基化修饰试剂盒 货号：A-R5001
EZ RNA Methylation Kit
背景资料：The EZ RNA Methylation™ Kit features rapid and reliable bisulfite treatment and conversion of cytosines in RNA for methylation analysis. The kit streamlines the three-step process for complete conversion of cytosine in into uracil. RNA denaturation and bisulfite conversion processes are combined into a single step. No buffer preparation is necessary. The RNA Conversion Reagent is provided ready-to-use: simply add the reagent to an RNA sample and incubate as indicated.Also, innovative in-column desulphonation technology eliminates messy precipitation steps, ensuring researchers obtain consistent results. The product has been designed to minimize template degradation, loss of RNA during treatment and clean-up, and to provide complete conversion of cytosine for accurate methylation analysis. Recovered RNA is ideal for RT-PCR, sequencing, library preparation and Next-Gen sequencing.
产品描述：
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RNA Input
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Samples containing 32 ng - 3 μg of DNA-free RNA. For optimal results, the amount of input RNA should be between 0.5 - 1 μg. |
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Conversion Efficiency
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> 99% of non-methylated C residues are converted to U with > 99% protection of 5-methylcytosine. |
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RNA Recovery
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> 80% |
步骤流程/测序分析：
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图示：EZ RNA甲基化修饰试剂盒的步骤
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经亚硫酸盐处理后测序结果. RNA甲基化C(5-mC)在核苷酸位点# 7使用EZ RNA甲基化修饰试剂盒工具处理。它经RT-PCR、克隆和测序,然后扩增，回收RNA。在位点# 7甲基化胞嘧啶无变化而在# 3、4和13非甲基化胞嘧啶完全转化为尿嘧啶(U)和通过RT-PCR和测序检测到胸腺嘧啶(T)。
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产品简易操作指南：
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Step 1
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Add 130 µl of RNA Conversion Reagent to 20 µl of RNA sample in a PCR tube. Mix the sample by flicking the tube or pipetting up and down, then centrifuge briefly to ensure there are no droplets in the cap or sides of the tube.
Note: If the sample volume is less than 20 µl, compensate with DNase/RNase-Free Water. |
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Step 2
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Place the PCR tube(s) in a thermal cycler and perform the following steps:
1. 70°C for 5 minutes
2. 54°C for 45 minutes
3. 4°C up to 20 hours Note: The 4°C storage step is optional.
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Step 3
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Place a Zymo-Spin™ IC Column into a Collection Tube and add 250 µl of RNA Binding Buffer to the column. |
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Step 4
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Load the sample (from Step 2) into the Zymo-Spin™ IC Column containing the RNA Binding Buffer and mix by pipetting up and down. |
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Step 5
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Add 400 µl of 95-100% ethanol to the sample-RNA Binding Buffer mixture in the column. Close the cap and immediately mix by inverting the column several times. |
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Step 6
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Centrifuge at full speed (≥10,000 x g) for 30 seconds. Discard the flow-through. |
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Step 7
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Add 200 µl RNA Wash Buffer to the column and centrifuge at full speed for 30 seconds. |
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Step 8
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Add 200 µl of RNA Desulphonation Buffer to the column and let stand at room temperature (20°C – 30°C) for 30 minutes. After the incubation, centrifuge at full speed for 30 seconds. Discard the flow-through. |
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Step 9
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Add 400 µl RNA Wash Buffer to the column and centrifuge at full speed for 30 seconds. Repeat the wash step with an additional 400 µl RNA Wash Buffer. |
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Step 10
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Centrifuge the Zymo-Spin™ IC Column in an emptied Collection Tube at full speed for 2 minutes. Remove the Zymo-Spin™IC Column carefully from the Collection Tube and transfer it into an RNase-free Tube. |
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Step 11
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Add ≥10 µl of DNase/RNase-Free Water directly to the column matrix and let stand for 1 minute at room temperature. Centrifuge at full speed for 30 seconds. The eluted RNA can be used immediately or stored at -20°C for up to 3 months. For long-term storage, keep at or below -70°C. |
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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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
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