基本描述

别名

miRNA qPCR试剂盒 | miRNA qPCR 检测试剂盒

英文名称

miRNA qPCR Assay Kit

储存温度

-20°C储存,避免反复冻融

运输条件

超低温冰袋运输

产品介绍

产品内容：

M665794	Component	125 T	Storage
M665794A	2×miRNA qPCR Mixture (ROX)	2×750 μL	-20℃. Avoid freeze/thaw cycle
M665794B	Reverse Primer, 10 μM	60 μL	-20℃. Avoid freeze/thaw cycle
M665794C	ddH2O	1.5 mL	-20℃. Avoid freeze/thaw cycle

产品简介：

本试剂盒采用SYBR Green I嵌合荧光染料法的原理来进行miRNA荧光定量PCR检测。试剂盒包括检测所需的2×miRNA qPCR Mixture和Reverse Primer。 2×miRNA qPCR Mixture是专门为miRNA定量检测而研发的新一代预混形式的荧光定量PCR检测试剂，所含的荧光染料SYBR Green I可以与所有的双链DNA结合，使该产品可用于不同靶序列的检测而不需合成特异性标记探针。其中的GoldStar Taq DNA polymerase是经化学修饰的高效热启动酶，配合独特的缓冲体系，使反应特异性更好，灵敏度更高，并能在更广的范围内对miRNA进行准确定量。2×miRNA qPCR Mixture含有ROX染料，适用于需要ROX作为校正染料的荧光定量PCR仪。

备注：本试剂盒须与miRNA cDNA第一链合成试剂盒配套使用。

自备实验材料：qPCR上游引物（Forward primer）。

Forward Primer设计原则：

1．遵循引物设计的最普遍原则。

2．以成熟的miRNA序列为基础，将U替换成T，这是最基础和最简单的设计方法。

3．试剂盒中提供的下游引物的Tm值为63.6℃，设计上游引物的Tm值要尽量保证在 63.6℃左右。

4．若按照原则“2”的方式直接设计的引物其Tm值过低，可以在引物的5’端添加几个碱基（最好为G或C碱基）；也可以在3’端添加1个或几个A碱基；或者5’端和3’端同时修饰。

5．若按照原则“2”的方式直接设计的引物其Tm值过高，可以在引物的5’或3’端去掉几个碱基。

注意事项：

1．试剂使用前请上下颠倒轻轻混匀，尽量避免起泡，并经短暂离心后使用。

2．miRNA 第一链cDNA 的加入量不要超过Real time PCR 体积10%。

3．对于特殊的检测体系，高含量的cDNA模板易导致非特异性扩增，根据所检测miRNA 的丰度适当的稀释cDNA（稀释10倍或者100倍）。

4．本产品中的2×miRNA qPCR Mixture中含有SYBR Green I和ROX染料，保存本产品或配制PCR反应液时应避免强光照射。

5．避免反复冻融本品，反复冻融可能使产品性能下降，本产品长期保存可置于-20℃。如果在短期内需要频繁使用，2×miRNA qPCR Mixture可于2-8℃保存。而Reverse primer仍需置于-20℃保存。

操作步骤：

1．室温融化2×miRNA qPCR Mixture和Reverse primer（10 μM）。

2．使用时请将2×miRNA qPCR Mixture上下颠倒轻轻均匀混合，避免起泡，并经短暂离心后使用。如果试剂没有混匀，其反应性能会有所下降。

3．将试剂置于冰上，并按下表配制反应体系：

试剂	体积	终浓度
2×miRNA-qPCR混合物（ROX）	10 μl	1×
正向素数，10μM	0.4μl	0.2 μM
反向引物（10 μM）	0.4μl	0.2 μM
miRNA第一链cDNA	X μl	—
ddH2O	高达20μl	—

4．反应程序设置如下：

注意！本产品预变性反应必须在95℃ 10分钟下完成！

注意：

1）本产品所采用的热启动酶须在预变性95℃、10 min条件下实现酶的活化。

2）退火温度请以60-64℃作为设定范围的参考，发生非特异性反应时，可提高退火温度。

Product content:

M665794	Component	125 T	Storage
M665794A	2×miRNA qPCR Mixture (ROX)	2×750 μL	-20℃. Avoid freeze/thaw cycle
M665794B	Reverse Primer, 10 μM	60 μL	-20℃. Avoid freeze/thaw cycle
M665794C	ddH2O	1.5 mL	-20℃. Avoid freeze/thaw cycle

Product Introduction:
This kit uses the principle of SYBR Green I chimeric fluorescent dye method for miRNA fluorescence quantitative PCR detection. The kit includes 2 x miRNA qPCR Mixture and Reverse Primer required for detection. 2 x miRNA qPCR Mixture is a new generation pre mixed form of fluorescence quantitative PCR detection reagent specially developed for miRNA quantitative detection. The fluorescent dye SYBR Green I contained in it can bind to all double stranded DNA, making the product suitable for detecting different target sequences without the need to synthesize specific labeled probes. The GoldStar Taq DNA polymerase is a chemically modified and highly efficient thermal starter enzyme, coupled with a unique buffer system, which enhances reaction specificity, sensitivity, and enables accurate quantification of miRNA over a wider range. The 2x miRNA qPCR Mixture contains ROX dye and is suitable for fluorescence quantitative PCR instruments that require ROX as a calibration dye.
Note: This kit must be used in conjunction with the miRNA cDNA first strand synthesis kit.
Self prepared experimental materials: qPCR upstream primer.

Forward Primer design principles:
1. Follow the most common principles of primer design.
2.Based on mature miRNA sequences, replacing U with T is the most basic and simplest design method.
3.The Tm value of the downstream primer provided in the reagent kit is 63.6 ℃, and the Tm value of the upstream primer should be designed to be around 63.6 ℃ as much as possible.
4. If the Tm value of the primer directly designed according to principle "2" is too low, several bases (preferably G or C bases) can be added to the 5 'end of the primer; One or several A bases can also be added at the 3 'end; Alternatively, both the 5 'and 3' ends can be modified simultaneously.
5.If the Tm value of a primer designed directly according to principle "2" is too high, several bases can be removed from the 5 'or 3' end of the primer.

Notes:
1. Before using the reagent, please gently mix it upside down to avoid foaming, and use it after a brief centrifugation.
2. The amount of miRNA first strand cDNA added should not exceed 10% of the volume of Real time PCR.
3. For special detection systems, high content of cDNA templates can easily lead to non-specific amplification. Dilute cDNA appropriately (10 or 100 times dilution) based on the abundance of detected miRNAs.
4. The 2x miRNA qPCR Mixture in this product contains SYBR Green I and ROX dyes. When storing this product or preparing PCR reaction solution, strong light exposure should be avoided.
5. Avoid repeated freezing and thawing of this product. Repeated freezing and thawing may cause a decrease in product performance. This product can be stored at -20 ℃ for long-term storage. If frequent use is required in the short term, the 2xmiRNA qPCR Mixture can be stored at 2-8 ℃. However, the Reverse primer still needs to be stored at -20 ℃.

Operation steps:
1. Melt 2 x miRNA qPCR Mixture and Reverse Primer at room temperature (10 μ M) .
2. When using, please gently mix the 2x miRNA qPCR Mixture upside down to avoid foaming, and use after brief centrifugation. If the reagent is not well mixed, its reaction performance will decrease.
3. Place the reagent on ice and prepare the reaction system according to the following table:

reagent	volume	final concentration
2×miRNA qPCR Mixture（ROX）	10 μl	1×
Forward primer（10 μM）	0.4μl	0.2 μM
Reverse primer（10 μM）	0.4μl	0.2 μM
MiRNA first strand cDNA	X μl	—
ddH2O	up to 20 μl	—

4. The reaction program is set as follows:
Attention!The pre denaturation reaction of this product must be completed at 95 ℃ for 10 minutes!

Note:

1) The hot start enzyme used in this product must be activated under pre denaturation conditions of 95 ℃ and 10 minutes.
2) The annealing temperature should be set at 60-64 ℃ as a reference range. When non-specific reactions occur, the annealing temperature can be increased.

质检证书（CoA,COO,BSE/TSE 和分析图谱)

C of A & Other Certificates(BSE/TSE, COO):

输入批号以搜索分析图谱:

技术文档和文章

m665794_mirna qpcr assay kit.pdf

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