GoldStar Probe One Step RT-qPCR Kit

产品内容

产品简介

本产品是采用探针法（TaqMan，Molecular Beacon等）进行一步法Real-Time RT- qPCR的专用试剂盒。使用本产品进行Real Time RT-qPCR反应时，逆转录和定量PCR 在同一反应体系中进行，反应过程中无需添加试剂，无需打开管盖，避免了污染的同时提高了实验效率。本产品的检测灵敏度高，荧光信号强，信噪比高，非常适合于RNA病毒等微量RNA的检测。其所包含的特殊缓冲系统能使逆转录酶与DNA聚合酶同时发挥最大功效，提高反应效率。使用本产品可以得到更宽广的线性范围，对目的基因定量更准确，重复性好，可信度高。

ROX染料用于校正定量PCR仪孔与孔之间产生的荧光信号误差，一般用于ABI、Stratagene等公司的Real Time PCR 扩增仪。不同仪器的激发光学系统有所不同，因此ROX染料的浓度必须与相应的荧光定量PCR仪相匹配。

不需要ROX校正的仪器（G665836） :

Roche LightCycler 480，Roche LightCyler 96，Bio-rad iCyler iQ，iQ5，CFX96等。

需要High ROX校正的仪器（G665801） ：

ABI Prism7000/7300/7700/7900，Eppendorf，ABI Step One/Step One Plus等。

注意事项

1．本试剂盒中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．本产品以RNA为模板进行一步法RT-PCR实验，在操作过程中应避免RNase污染， 建议在专门的区域进行RNA操作，使用专门的仪器和耗材，操作人员带口罩和一次 性手套并经常更换手套，实验相关耗材应用0.1％DEPC（焦碳酸二乙酯）水溶液在 37 ℃处理12小时,并高压灭菌30分钟后使用。 

3．本试剂盒中的各试剂应尽量避免反复冻融，反复冻融可能使产品性能下降。 

4．本试剂盒必须使用特异性引物，引物的选择可根据具体实验来选择，引物设计的好 坏直接影响到RT-qPCR反应的结果，设计引物时需考虑GC含量，引物长度，引物 位置，PCR产物的二级结构等因素，建议采用专业的引物设计软件进行设计。 

5．本试剂盒推荐使用特异性探针，建议采用专业的设计软件进行设计。

使用方法

以下举例为常规的反应体系和反应条件，实际操作中应根据模板、引物结构和目的片 段大小的不同进行相应的改进和优化。（反应液的配置请在冰上进行） 

1．将RNA模板、引物、2×GoldStar Probe One Step Buffer、GoldStar Probe One Step EnzymeMix和RNase-Free Water溶解并置于冰上备用。 

2．PCR反应体系：

注意：1）通常引物浓度以0.2 μM可以得到较好结果，可以在0.1-1.0 μM作为设定范围的参考。 

2）使用的探针浓度，与使用的荧光定量PCR仪、探针种类、荧光标记物质种类有关，实际使 用时请参照仪器说明书，或各荧光探针的具体使用要求进行浓度的调节。 

3）通常RNA模板的量以10 pg – 100 ng为参照，因不同物种的模板中含有的目的基因拷贝数不 同，可对模板进行梯度稀释，以确定最佳的模板使用量。 

4）不同仪器的激发光学系统有所不同，根据使用荧光定量的仪器选择加入50×Low ROX or 50× High ROX。

3．混匀，短暂离心，将溶液收集到管底。 

4．RT-PCR反应条件：

注意：1）本产品所采用的热启动酶须在预变性95℃、5-10 min条件下实现酶的活化。 

2）建议采用两步法PCR反应程序，若因使用Tm值较低的引物等原因，得不到良好的实验结果时，可尝试进行三步法PCR扩增，退火温度请以 56℃-64℃的范围作为设定参考。

Product content

Product Introduction

This product is a specialized kit for one-step Real-Time RT-qPCR using the probe method (TaqMan, Molecular Beacon, etc.). When using this product for Real Time RT-qPCR reaction, reverse transcription and quantitative PCR are carried out in the same reaction system, and there is no need to add reagents or open the cap of the tube during the reaction process, which avoids contamination and improves the experimental efficiency at the same time. With high detection sensitivity, strong fluorescence signal and high signal-to-noise ratio, this product is very suitable for the detection of RNA viruses and other trace RNA. The special buffer system contained in this product can maximize the effectiveness of reverse transcriptase and DNA polymerase at the same time and improve the efficiency of the reaction. A wider linear range can be obtained with this product, more accurate quantification of the target gene, good reproducibility and high confidence.

ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.

 Instruments that do not require ROX calibration (G665836)

Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others.

 Instruments that require High ROX calibration (G665801)  

ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.

matters needing attention

1.Before using the reagents in this kit, please mix them gently by turning them up and down to avoid foaming as much as possible, and use them after brief centrifugation.

2.This product uses RNA as the template for one-step RT-PCR experiment, RNase contamination should be avoided during operation, it is recommended to operate RNA in a special area, use special instruments and consumables, the operator with a mask and disposable gloves and often change the gloves, the experiment-related consumables should be processed with 0.1% DEPC (diethyl ether pyrocarbonate) aqueous solution for 12 hours at 37℃, and autoclaved for 30 minutes before use. The consumables should be treated with 0.1% DEPC (diethylpyrocarbonate) aqueous solution at 37℃ for 12 hours and autoclaved for 30 minutes.

3.Repeated freezing and thawing of each reagent in this kit should be avoided as much as possible; repeated freezing and thawing may degrade the product performance.

4.This kit must use specific primers, the choice of primers can be selected according to specific experiments, the good or bad primer design directly affects the results of RT-qPCR reaction, the design of primers need to consider the GC content, primer length, primer position, the secondary structure of the PCR product and other factors, it is recommended to use a professional primer design software for design.

5.This kit is recommended to use specific probes, and it is recommended to use professional design software for designing.

Usage

The following examples are conventional reaction systems and conditions, which should be improved and optimized according to the different templates, primer structures and target fragment sizes in actual operation. (Please prepare the reaction solution on ice.)

1. Dissolve RNA template, primers, 2× GoldStar Probe One Step Buffer, GoldStar Probe One Step EnzymeMix and RNase-Free Water and set aside on ice.

2. PCR reaction system:

Note: 1) Usually, better results can be obtained with a primer concentration of 0.2 μM, and 0.1-1.0 μM can be used as a reference for setting the range.

(2) The concentration of the probe used is related to the fluorescence quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, please refer to the instrument manual or the specific requirements for the use of each fluorescent probe for the adjustment of the concentration in actual use.

(3) Usually the amount of RNA template is 10pg-100ng as a reference. Since the templates of different species contain different copy numbers of target genes, the templates can be diluted in gradient to determine the optimal amount of template to use.

(4) The excitation optical system varies from instrument to instrument, choose to add 50×Low ROX or 50×High ROX according to the instrument using fluorescence quantification.

3. Mix well, centrifuge briefly, and collect the solution at the bottom of the tube.

4.RT-PCR reaction conditions:

Note: 1) The hot start enzyme used in this product must be activated under the condition of pre-denaturation 95℃, 5-10min. 2) It is recommended to use the two-step PCR reaction program, if you can not get good experimental results due to the use of primers with lower Tm value, etc., you can try to carry out the three-step PCR amplification, and the annealing temperature should be set in the range of 56℃-64℃ as a reference.