产品描述

Hieff^® qPCR SYBR Green Master Mix是2×实时定量PCR扩增的预混合溶液。Mix中含有热启动Hieff^® DNA Polymerase、SYBR Green I、dNTPs、Mg2+。使用时，仅需在扩增体系中加入模板和引物即可进行实时荧光定量PCR，大大简化操作过程，降低污染几率。本产品针对不同型号的实时荧光定量PCR仪，分别提供不同浓度的 Rox 参比液（High Rox/Low Rox），用于校正孔与孔之间的荧光信号误差。

本品采用的DNA聚合酶配体可以随温度变化实时调节DNA聚合酶活性。配方添加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，使定量PCR可以在宽广的定量区域内获得良好的线性关系。

产品组分

运输与保存方式

冰袋运输。-20℃避光储存，有效期18个月。

本品避免反复冻融。产品中含有荧光染料SYBR® Green I，保存或配制反应体系时需避免强光照射。

注意事项

1. 推荐使用本公司cDNA合成试剂盒（货号：11123ES），以有效去除RNA样品中残留的基因组。

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！

反应体系（推荐冰上配制）

【注】： 使用前务必充分混匀，避免剧烈震荡产生过多气泡。

a) 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】。

b) 引物浓度：通常引物终浓度为0.2 μM，也可以根据情况在0.1-1.0 μM之间进行调整。

c) 模板浓度：如模板类型为未稀释cDNA原液，使用体积不应超过qPCR反应总体积的1/10。

d) 模板稀释：cDNA原液建议5-10倍稀释，最佳模板加入量以扩增得到的CT值在20-30个循环为好。

e) 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。

f) 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。

扩增程序

三步法程序                                                                                                 两步法程序

【注】：高特异性可选择两步法，高效率扩增可选择三步法。

a) 预变性时间：根据不同模板和引物的具体情况可适当缩短至2 min。

b) 退火温度和时间：请根据引物和目的基因的长度进行调整。

c) 荧光信号采集（★）：请参考仪器说明书设置。 

d) 熔解曲线：通常情况下可以使用仪器默认程序。

结果分析

定量实验至少需要三个生物学重复。反应结束后需要确认扩增曲线及熔解曲线。

1) 扩增曲线：标准扩增曲线为S型。

Ct值落在20-30之间时，定量分析最准确；

Ct值小于10，需要将稀释模板后，重新进行实验；

Ct值介于30-35之间时，需要提高模板浓度，或者增大反应体系的体积，以提高扩增效率，保证结果分析的准确性；

Ct值大于35时，检测结果无法定量分析基因的表达量，但可用于定性分析。

2) 熔解曲线：

熔解曲线单峰，表明反应特异性好可以进行定量结果分析；若熔解曲线出现双峰或者多峰，则不能进行定量分析。

熔解曲线出现双峰，需要通过DNA琼脂糖凝胶电泳判断非目标峰是引物二聚体还是非特异性扩增。

如果是引物二聚体，建议降低引物浓度，或者重新设计扩增效率高的引物。

如果是非特异性扩增，请提高退火温度，或者重新设计更高特异性的引物。

引物设计指南

1）推荐引物长度25 bp左右。扩增产物长度150 bp为佳，可以在100 bp-300 bp内选择。

2）正向引物和反向引物的Tm值相差不宜超过2℃。引物Tm值60ºC-65ºC为佳。

3）引物碱基分布要均匀，避免出现连续的4个相同碱基，GC含量控制在50%左右。3’端最后一个碱基最好为G或C。

4）引物内部或者正反两条引物间最好避免出现有3个碱基以上的互补序列。

5）引物特异性需要用NCBI BLAST程序进行核对。避免引物3’端有2个碱基以上的非特异性互补。

6）设计完成的引物需要进行扩增效率的检测，只有具备相同扩增效率的引物才可用于定量比较分析。

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, QuantStudio6,7,12k 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, LightCycler 2.0; Lightcycler 96; Thermo Scientific PikoReal Cycler;

Cepheid SmartCycler; Illumina Eco qPCR. 

相关产品

HB220113

Q：模板用量X 是多少？常用的量是多少？

A：a)X 表示模板 DNA 量需要实验者在首次实验时进行摸索。首先对模板DNA 进行稀释（一般推荐 5-10 倍），然后模板量梯度上样，选择 CT 值落在  20-30  之间的最佳上样量。

b）常用的量是逆转录 500-1000ng 总RNA，稀释 10 倍取 1μL cDNA 进行qPCR 实验。

Q：qPCR 实验结果的有效性？为什么建议Ct 值要大于 15？

A：a)有效性要满足三个条件：

（1）标准曲线：扩增效率范围:90-110%，对应斜率为-3–3.5。R2＞0.98。 (扩增效率=10-1/斜率-1)，当斜率=-3.32 时，扩增效率=100%。

（2）扩增曲线：S 型曲线，且 Ct 值在 15－35 之间，阴性对照 Ct＞35 或无Ct 值。

（3） 熔解曲线：为单一峰。

b)3-15 个循环的荧光值标准差的 10 倍是荧光阈值，Ct 值太小了会影响曲线。

Q：Rox 的作用？

A：ROX 是一种参比染料，其作用是标准化荧光定量反应中的非PCR 震荡，校正加样误差或者是孔与孔之间的误差，提供一个稳定的基线。

Q：为什么扩增曲线不稳定（扩增曲线平台期锯齿状）？

A：可能原因：

a）RNA 纯度低，体系中存在较多杂质；推荐参数：OD260/OD280=1.8-2.0， OD260/OD230＞2.0。随着 qPCR 反应的进行，阻碍反应的因素不断增加，若 RNA 纯度低，杂质多，会进一步影响仪器平台期的算法，导致出现锯齿状。

b）仪器长时间未做校准。仪器未校准会使仪器算法错误，导致各种异常结果。

解决方案：

a）先梯度加大模板稀释倍数看优化效果。若效果仍不好建议新制备高纯度RNA 重新实验。b）定期（一般 1 年）进行仪器校准保养。

Q：为什么扩增曲线无法达到平台期？

A：可能原因：

a）模板量太低（CT 值 35 左右）。推荐 Ct 值：15＜Ct＜30。原因：Ct 值太大（如  Ct＞30），刚进入指数扩增期几个循环就停止了反应，故无法到达平台期。

b）循环次数太少（30 cycles）；循环次数过少（如 35）导致刚进入指数扩增期几个循环就停止了反应，故无法到达平台期。

c）试剂扩增效率低（CT 小，但无法达到平台期，曲线比较“趴”）。

解决方案：

a）提高模板量；参考 Q1 的优化方法。

b）提高循环次数；推荐循环数：一般 40。低丰度基因可设 45。

c）做标准曲线测定扩增效率，若确实偏低，则换试剂。

d）增加 Mg2+浓度（会增加非特异扩增）

Q：为什么出现双峰，并且较低峰 Tm 在 80℃之前？

A：较低峰 Tm 在 80℃之前可能原因：存在引物二聚体（一般mRNA 反转录后定量，产物在 100-150bp 左右，峰对应 Tm 值为 80-90℃。若有引物二聚体存在，引物二聚体大小只有几十bp，峰对应 Tm 值在 70-80℃之间。故会在 80℃以前出现一个峰， 80℃以后出现一个峰），模板浓度过低或引物浓度过高。

解决方案：

a）适当提高退火温度； b）提高模板量，降低引物浓度； c）重新设计引物。

Q：为什么出现双峰，双峰 Tm 都在 80℃以前？

A：双峰Tm 都在 80℃以前的可能原因：做 MicroRNA 定量时存在引物二聚体。Micro RNA 反转录后定量， 产物在 80-90bp 左右，峰对应Tm 值为 70-80℃，若有引物二聚体存在，引物二聚体大小只有几十 bp，峰对应 Tm 值也在 70-80℃之间。故会在 80℃ 以前出现双峰。

优化方法：提高退火温度、降低引物浓度或重新设计引物等方式优化。

Q：为什么出现双峰，并且双峰Tm 都在 80℃以后？

A：可能原因：

a）引物特异性过差导致非特异性产物扩增。

b）交叉污染。

c）gDNA 污染，可通过 NRC 进行确认。

解决方案：

a）Blast 检查引物特异性，差则重新设计引物。

b）超净台中操作，注意更换 Tip 头，避免交叉污染。

c）通过 NRC 阴性对照进行确认，若有，需重新制备模板。

Q：为什么是单峰，但 Tm 在 80℃之前？

A：可能原因：

扩增产物是完全的引物二聚体，可能是未加模板。

注：若 microRNA，则结果正常（做 microRNA 定量时存在引物二聚体。micro RNA 反转录后定量， 产物在 80-90bp 左右，峰对应 Tm 值为 70-80℃，若有引物二聚体存在，引物二聚体大小只有几十bp，峰对应 Tm 值也在 70-80℃之间。故会在 80℃以前出现双峰）。

解决方案：

进行高分辨率琼脂糖电泳，检测有无目的条带，以确定模板是否加入。优化方法：新配制无误的反应体系，重新实验。

Q：为什么是单峰，但峰不尖锐？

A：可能原因：

存在大小相近的非特异性扩增

解决方案：a）温度跨度不高于 7℃，视为可用结果（即 Tm 值跨度＜7℃可认为是同一种产物）；

b）进行高浓度琼脂糖电泳（高分辨率），确认是否为单一条带。

1. Lu, XY., Shi, XJ., Hu, A. et al. Feeding induces cholesterol biosynthesis via the mTORC1–USP20–HMGCR axis. Nature (2020). （IF43）

2. Han X., et al., Mapping the Mouse Cell Atlas by Microwell-Seq[J]. Cell. 2018 Feb 22;172(5):1091-1107.e17. （IF 30.410）

3. Wang X, Ni L, Wan S, et al. Febrile temperature critically controls the differentiation and pathogenicity of T helper 17 cells[J]. Immunity, 2020, 52(2): 328-341. e5. （IF21.522）

4. Xiao J, Li W, Zheng X, et al. Targeting 7-Dehydrocholesterol Reductase Integrates Cholesterol Metabolism and IRF3 Activation to Eliminate Infection[J]. Immunity, 2020, 52(1): 109-122. e6.（IF21.522）

5. Han F, Liu X, Chen C, et al. Hypercholesterolemia risk-associated GPR146 is an orphan G-protein coupled receptor that regulates blood cholesterol levels in humans and mice[J]. Cell Research, 2020, 30(4): 363-365.（IF20.5）

6. Mo J, Chen Z, Qin S, et al. TRADES: Targeted RNA Demethylation by SunTag System[J]. Advanced Science, 2020, 7(19): 2001402.（IF15.8）

7. Fan H, Hong B, Luo Y, et al. The effect of whey protein on viral infection and replication of SARS-CoV-2 and pangolin coronavirus in vitro[J]. Signal transduction and targeted therapy, 2020, 5(1): 1-3.(IF13.493)

8. Wang J., et al., The mycobacterial phosphatase PtpA regulates the expression of host genes and promotes cell proliferation[J]. Nat Commun. 2017 Aug 15;8(1):244. （IF 12.353）

9. Zhou L, Hou B, Wang D, et al. Engineering Polymeric Prodrug Nanoplatform for Vaccination Immunotherapy of Cancer[J]. Nano Letters, 2020.（IF12.279）

10. Wang Y, Xiao Y, Dong S, et al. Antibody-free enzyme-assisted chemical approach for detection of N 6-methyladenosine[J]. Nature Chemical Biology, 2020: 1-8.(IF12.154)

11. Zhu M, Dai X. Maintenance of translational elongation rate underlies the survival of Escherichia coli during oxidative stress[J]. Nucleic acids research, 2019.（IF11.6）

12. Su G, Guo D, Chen J, et al. A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation[J]. Nucleic acids research, 2019.（IF11.6）

13. Feng Y, Wang Y, Wang X, et al. Simultaneous Epigenetic Perturbation and Genome Imaging Reveal Distinct Roles of H3K9me3 in Chromatin Architecture and Transcription[J]. bioRxiv, 2020.（IF10.806 ）

14. Huang X, He M, Huang S, et al. Circular RNA circERBB2 promotes gallbladder cancer progression by regulating PA2G4-dependent rDNA transcription[J]. Molecular cancer, 2019, 18(1): 166.（IF10.679）

15. Wang Z, Liu C, Zhu D, et al. Untangling the co-effects of oriented nanotopography and sustained anticoagulation in a biomimetic intima on neovessel remodeling[J]. Biomaterials, 2020, 231: 119654.(IF10.317)

16. Wang Z, Liu C, Zhu D, et al. Untangling the co-effects of oriented nanotopography and sustained anticoagulation in a biomimetic intima on neovessel remodeling[J]. Biomaterials, 2019: 119654.（IF10.273）

17. Yafen Wang, Xiong Zhang, et al. Bisulfite-free, single base-resolution analysis of 5-hydroxymethylcytosine in genomic DNA by chemical-mediated mismatch[J]. Chem. Sci., 2019, Advance Article. （IF 9.063）

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产品描述

Hieff® qPCR SYBR Green Master Mix(No Rox)是2×实时定量PCR扩增的预混合溶液。Mix中含有热启动Hieff^® DNA Polymerase、SYBR Green I、dNTPs、Mg²⁺。使用时，仅需在扩增体系中加入模板和引物即可进行实时荧光定量PCR，大大简化操作过程，降低污染几率。

本品采用的DNA聚合酶配体可以随温度变化实时调节DNA聚合酶活性。配方添加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，使定量PCR可以在宽广的定量区域内获得良好的线性关系。

适用机型

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, LightCycler 2.0; Lightcycler 96;

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

运输和保存方式

冰袋运输。-20℃避光储存，有效期18个月。

本品避免反复冻融。产品中含有荧光染料SYBR Green I，保存或配制反应体系时需避免强光照射。

注意事项

1. 推荐使用本公司cDNA合成试剂盒（货号：11123ES），以有效去除RNA样品中残留的基因组。

2. 解冻后Master Mix可能出现絮状物质，4℃放置并上下颠倒混匀至溶液澄清，不影响试剂性能。

3. 为了您的安全和健康，请穿实验服并佩戴一次性手套操作。

4. 本产品仅作科研用途！

反应体系（推荐冰上配制）

【注】： 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 μM，也可以根据情况在0.1-1.0 μM之间进行调整。

b) 模板浓度：如模板类型为未稀释cDNA原液，使用体积不应超过qPCR反应总体积的1/10。

c) 模板稀释：cDNA原液建议5-10倍稀释，最佳模板加入量以扩增得到的CT值在20-30个循环为好。

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。
 

【注】: 高特异性可选择两步法，高效率扩增可选择三步法。

a) 预变性时间：根据不同模板和引物的具体情况可适当缩短至2 min。

b) 退火温度和时间：请根据引物和目的基因的长度进行调整。

c) 荧光信号采集（^★）：请按照仪器使用说明书要求进行实验程序设置，几种常见仪器的时间设定如下：

30sec以上：Applied Biosystems: StepOne, StepOne Plus, 7500 Fast；Roche Applied Science: LightCycler 480；Bio-Rad: CFX96

31sec以上：Applied Biosystems: 7300

34sec以上：Applied Biosystems: 7500

d) 熔解曲线：通常情况下可以使用仪器默认程序。

结果分析

定量实验至少需要三个生物学重复。反应结束后需要确认扩增曲线及熔解曲线。

1) 扩增曲线：标准扩增曲线为S型。

Ct值落在20-30之间时，定量分析最准确；

Ct值小于10时，需要将稀释模板后，重新进行实验；

Ct值介于30-35之间时，需要提高模板浓度，或者增大反应体系的体积，以提高扩增效率，保证结果分析的准确性；

Ct值大于35时，检测结果无法定量分析基因的表达量，但可用于定性分析。

2) 熔解曲线：

熔解曲线单峰，表明反应特异性好可以进行定量结果分析；若熔解曲线出现双峰或者多峰，则不能进行定量分析。

熔解曲线出现双峰，需要通过DNA琼脂糖凝胶电泳判断非目标峰是引物二聚体还是非特异性扩增。

如果是引物二聚体，建议降低引物浓度，或者重新设计扩增效率高的引物。

如果是非特异性扩增，请提高退火温度，或者重新设计更高特异性的引物。

引物设计指南

1. 推荐引物长度25 bp左右。扩增产物长度150 bp为佳，可以在100 bp-300 bp内选择。

2. 正向引物和反向引物的Tm值相差不宜超过2℃。引物Tm值60℃-65℃为佳。

3. 引物碱基分布要均匀，避免出现连续的4个相同碱基，GC含量控制在50%左右。3’端最后一个碱基最好为G或C。

4. 引物内部或者正反两条引物间最好避免出现有3个碱基以上的互补序列。

5. 引物特异性需要用NCBI BLAST程序进行核对。避免引物3’端有2个碱基以上的非特异性互补。

6. 设计完成的引物需要进行扩增效率的检测，只有具备相同扩增效率的引物才可用于定量比较分析。

相关产品

HB210720

A：一般 CT 值与模板起始浓度呈负相关，浓度越高，CT 值越小。但也有很多特殊情况， 比如体系中存在抑制物或是模板不纯，这时候稀释模板反而能使 CT 值变低。

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Q：不加 ROX 的qPCR mix 后续需换用其他仪器（需要 ROX），是否可以单独加 ROX后再使用？

A：可以。

Q：可以用于microRNA 或lncRNA 等非编码RNA 吗？环状 RNA 呢？

A：不推荐。

Q：建议qPCR 实验用几步法？

A：常用 2 步法。需提高扩增特异性，可选用 2 步法或提高退火温度。在扩增效率低， ct 值过大的时候，可以改用 3 步法或延长延伸时间。

Q：预变性 5 min 调整成了 10min，对于实验结果有影响吗？

A：有影响，预变性时间较长可能会影响酶的活性，导致 PCR 产物的产量有所降低。

Q：qPCR 实验结果的有效性？为什么建议Ct 值要大于 15？

A：有效性要满足三个条件：（1）标准曲线：扩增效率范围:90–110%，对应斜率为  –3—3.5。 R2＞0.98。 (扩增效率=10-1/斜率–1)，当斜率=-3.32 时，扩增效率=100%。（2）扩 增曲线：S 型曲线，且 Ct 值在 15－35 之间，阴性对照 Ct＞35 或无 Ct 值。（3）熔解曲线：为单一峰。

Ct 值大于 15 个循环是因为 3-15 个循环的荧光值标准差的 10 倍是荧光阈值，Ct 值太小了会影响曲线。

Q：同一基因复孔间熔解曲线 Tm 值有差异？

A：同样的扩增产物也会出现Tm 值有微小差异，一般差异在 1 度以内都可以接受。

Q：为什么稀释了模板CT 值反而变小了？