Hifair® Ⅲ One Step RT-qPCR SYBR Green Kit是以RNA为模板进行定量PCR反应的试剂盒。在实验的过程中，逆转录和定量PCR在同一反应管中进行，简化了实验操作，降低了污染的风险。本试剂盒以RNA为模板，使用基因特异性引物，利用耐热Hifair® Ⅲ Reverse Transcriptase高效合成第一链cDNA，并以此为模板在UNICON® HotStart Taq DNA Polymerase作用下，配合优化的缓冲体系能够特异灵敏的对RNA模板进行精确定量。同时Buffer中额外添加了有效抑制非特异性PCR扩增的组分和提高扩增能力的促进因子，使定量PCR结果可以在宽广范围内更加真实有效。

本试剂盒以Kit形式提供，2×Hifair® Ⅲ SG buffer包含优化的缓冲体系和dNTPs等，优化了有效抑制非特异性PCR扩增的因子和提升PCR反应扩增效率的因子，适用于SYBR Green标记的高特异性检测系统。此外，Hifair® UH Ⅲ Enzymes包含比例优化的Hifair® Ⅲ Reverse Transcriptase、RNase inhibitor及UNICON® HotStart Taq DNA Polymerase等。

 

产品信息

货号	11143ES50 / 11143ES70 / 11143ES80
规格	50 T / 200 T / 500 T

 

组分信息

组分编号	组分名称	11143ES50	11143ES70	11143ES80
11143-A	2×Hifair® Ⅲ SG buffer*	500 μL	4×500 μL	5 mL
11143-B	Hifair® UH Ⅲ Enzymes	50 μL	200 μL	500 μL
11143-C	Hieff® 50×High Rox	50 μL	200 μL	500 μL
11143-D	Hieff® 50×Low Rox	50 μL	200 μL	500 μL
11143-E	RNase-free ddH2O	500 μL	4×500 μL	5 mL

*SG buffer为SYBR Green buffer的简写，UH为UNICON-Hifair的简写。

 

储存条件

-25~-15℃避光储存，有效期1年。本品避免反复冻融。建议分装保存。

 

使用说明

组分	体积 （μL）	终浓度
2×Hifair® Ⅲ SG buffer	10	1×
Hifair® UH Ⅲ Enzymes	1	–
Hieff® 50×High Rox or Low Roxb	0.4	1×
Forward Primer (10 μmol/L)	0.4	0.2 μmol/La
Reverse Primer (10 μmol/L)	0.4	0.2 μmol/La
模板 RNAc	Total RNA 1 pg-1 μg	–
RNase-free ddH2O	to 20d	–

表1反应体系(以20 μL为例)**

**使用前务必充分混匀，避免剧烈震荡产生过多气泡。

a. 引物浓度：通常引物终浓度为0.2 μmol/L，也可以根据情况在0.1-1.0 μmol/L之间进行调整。

b. 参比染料：Rox的添加，可根据不同仪器型号进行选择，具体可参考【适用机型】。

c. 模板稀释：qPCR灵敏度极高，建议将模板进行稀释使用，控制CT值在20-30之间适宜。

d. 反应体系：推荐使用20 μL或50 μL，以保证目的基因扩增的有效性和重复性。

e. 体系配制：请于超净工作台内配制，并使用无核酸酶残留的枪头、反应管；推荐使用带滤芯的枪头。避免交叉污染和气溶胶污染。

 

循环步骤	温度	时间	循环数
逆转录	42℃b	10 mina	1
预变性	95℃	5 min	1
扩增反应	95℃	10 sec	40
	60℃	30 sec
熔解曲线d	仪器默认设置		1

表2 标准扩增程序***

***针对实验需求可做适当调整。

a. 逆转录：对于一些极低丰度的基因可以将逆转录时间适当延长至15 min。

b. 温度：对于含复杂二级结构或高GC含量的RNA模板，可将逆转录温度提高到55℃，有助于提升灵敏度。

c. 荧光信号采集：不同的qPCR检测仪器所需的荧光信号采集时间不同，请根据最短时间限制进行设置。

d. 熔解曲线：通常情况下可以使用仪器默认程序。

 

适用机型

High Rox适用机型： ABI 5700, 7000, 7300, 7700, 7900HT Fast, StepOne, StepOne Plus;

Low Rox适用机型： ABI 7500, 7500 Fast, ViiA7, QuantStudio 3 and 5, QuantStudio 6, 7, 12k Flex;

     Stratagene MX3000P, MX3005P, MX4000P;

不需要Rox校正的仪器型号：

Bio-Rad: CFX96, CFX384, iCycler iQ, iQ5, MyiQ, MiniOpticon, Opticon, Opticon 2, Chromo4;

Eppendorf: Mastercycler ep realplex, realplex 2 s;

Qiagen: Corbett Rotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000;

Roche Applied Science: LightCycler 480, LightCycler2.0, Lightcycler 96;

Thermo Scientific: PikoReal Cycler; Cepheid: SmartCycler; Illumina: Eco qPCR.

 

注意事项

1. 实验过程中请使用RNase-free耗材。
2. 为了您的安全和健康，请穿实验服并佩戴一次性手套操作。
3. 本产品仅作科研用途！

 

Ver.CN20230404

Q：产品的应用领域和特点是什么？

A：应用于病毒检测，特点是高检出率

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