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本试剂盒采用在miRNA 3’末端加多聚A尾的方法来使miRNA具有Poly(A)尾，之后 再使用Oligo(dT)-Universal tag通用逆转录引物进行逆转录反应，最终合成miRNA对应 的**链cDNA。 　　miRNA cDNA**链合成试剂盒包含miRNA 3’末端Poly(A)尾修饰过程及修饰后逆 转录过程需要的全部试剂。该试剂盒具有非常高的Poly(A)修饰和逆转录效率，可以从 1 ng-2 μg的total RNA中有效获得miRNA对应的cDNA**链。并且操作简便、快捷， 可以用于从一个反应合成的cDNA中同时检测多种miRNA，不仅减少了误差、节约了样 品，同时还实现了检测的高通量。

备注：本试剂盒须与miRNA荧光定量检测试剂盒配套使用。   

自备实验材料：1 ng-2 μg的总 RNA，或0.1 ng-1 μg的小分子RNA。

注意事项：

预防RNase污染，应注意以下几方面： 

1．使用无RNase的塑料制品和枪头，避免交叉污染。 

2．玻璃器皿应在使用前于180℃高温下干烤4小时，塑料器皿可在0.5 M NaOH中浸泡 10分钟，用水彻底冲洗后高压灭菌。 

3．配制溶液应使用无RNase的水。 

4．操作人员戴一次性口罩和手套，实验过程中要勤换手套。  

使用方法：

A．miRNA加Poly(A)尾的过程： 

1．首先根据所用RNA的量，按如下公式，用1 mM Tris（PH8.0）来稀释10 mM ATP： ATP稀释系数=5000/__ng的总RNA 

例：如果总RNA的起始用量为100 ng，那么ATP稀释系数=5000/100=50。即将ATP 稀释50倍（1 μl的10 mM ATP加49 μl的1 mM Tris，PH8.0）。 

2．向冰浴中预冷的无RNase反应管中加入以下试剂至总体积25 μl。

*反应中使用的total RNA必须含有小分子RNA。

此过程也可以直接使用小分子RNA（建议加入量为2-5 μl。请根据目的miRNA的丰 度来确定加入量）。 

3．轻轻混匀上述反应液，短暂离心将液体收集于管底。37℃，孵育15分钟。此过程结 束后，立刻进行**链cDNA的合成，或于-20℃暂存。如需长期保存，建议存放于 -80℃。  

B．修饰后的miRNA cDNA**链合成的过程：

1．向冰浴中预冷的无RNase反应管中加入下表中试剂，至终体积20 μl：

2．轻轻混匀上述反应液，短暂离心将液体收集于管底。42℃，孵育50分钟。 

3．85℃，孵育5分钟，终止反应。合成的cDNA反应液可以直接进行荧光定量检测实 验，也可以于-20℃保存备用。  

Product content:

This kit uses the method of adding a poly (A) tail at the 3 'end of miRNA to give miRNA a Poly (A) tail, followed by reverse transcription using Oligo (dT) - Universal tag universal reverse transcription primers to synthesize the first stranded cDNA corresponding to miRNA. The miRNA cDNA first strand synthesis kit contains all the reagents required for the miRNA 3 'end Poly (A) tail modification process and the reverse transcription process after modification. This kit has a very high Poly (A) modification and reverse transcription efficiency, which can range from 1 ng-2 μ The first strand of cDNA corresponding to miRNA was effectively obtained from the total RNA of g. And the operation is simple and fast, which can be used to simultaneously detect multiple miRNAs from a synthesized cDNA reaction. This not only reduces errors and saves samples, but also achieves high-throughput detection.
Note: This kit must be used in conjunction with the miRNA fluorescence quantitative detection kit.
Self prepared experimental materials: 1 ng-2 μ Total RNA of g, or 0.1 ng-1 μ Small molecule RNA of g.

Notes:
To prevent RNase pollution, attention should be paid to the following aspects:
1. Use plastic products and gun heads without RNase to avoid cross contamination.
2. Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use. Plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.
3. The solution should be prepared using water without RNase.
4. Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.

Usage:
A. The process of miRNA adding Poly (A) tail:
1.based on the amount of RNA used, dilute the total RNA of 10 mM ATP with 1 mM Tris (pH 8.0) according to the following formula: ATP dilution coefficient=5000/__ ng
Example: If the initial amount of total RNA is 100 ng, then the ATP dilution coefficient is 5000/100=50. About to dilute ATP 50 times (1 μ 10 mM ATP plus 49 for l μ 1 mM Tris at pH 8.0.
2. Add the following reagents to the pre cooled RNase free reaction tube in the ice bath to a total volume of 25 μ L.

*The total RNA used in the reaction must contain small molecule RNA.

This process can also directly use small molecule RNA (recommended dosage of 2-5) μ L. Please determine the amount added based on the abundance of the target miRNA.
3. Gently mix the above reaction solution and briefly centrifuge to collect the liquid at the bottom of the tube. Incubate at 37 ℃ for 15 minutes. After this process is completed, immediately proceed with the synthesis of the first strand cDNA or temporarily store it at -20 ℃. If long-term storage is required, it is recommended to store at -80 ℃.
B. The process of synthesizing the first strand of modified miRNA cDNA:
1. Add the reagents in the table below to the pre cooled RNase free reaction tube in the ice bath until the final volume reaches 20μl:

2. Gently mix the above reaction solution and briefly centrifuge to collect the liquid at the bottom of the tube. Incubate at 42 ℃ for 50 minutes.
3.85 ℃ for 5 minutes and terminate the reaction. The synthesized cDNA reaction solution can be directly used for fluorescence quantitative detection experiments or stored at -20 ℃ for future use.