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干货 | 实验篇之荧光定量 PCR（上）

发布: 2019-01-09 2,035阅读 557评论

哈喽啊~小编又来了…了…了…作为一名认真的科研狗，今天要继续给大家分享科研道路上的干货，让大家少走弯路，直通成功！

首先，我们还是要一起复习一下何为荧光定量 PCR。荧光定量 PCR ( realtime fluorescence quantitative PCR，RTFQ PCR ）是一种定量试验技术，通过荧光染料或荧光标记的特异性的探针，对 PCR 产物进行标记跟踪，实时在线监控反应过程，结合相应的软件可以对产物进行分析，计算待测样品模板的初始浓度。

概念非常通俗易懂，但是科研道路上还是有许多无法跳过的关卡，曾让小编夜不能眠、食不下咽，借此小编将实验中遇到的问题进行了记录和整理，共享给大家。

Q：要把 qPCR 实验做，统共分几步？
A：三步！
Q：哪三步？
A：前期准备、中期操作、后期分析！

第三步会在下一专题和大家分享，今天先聊聊前两步：

一、qPCR 实验前期准备
前期准备主要是提取、反转录和引物设计部分，我们来逐一分享。

1. RNA 提取注意事项
首先了解一下，RNA 抽提原则：保证 RNA 的一级结构、无其它物质的污染、得率高、覆盖所有转录本。再比较一下 Trizol 提取和试剂盒提取，Trizol 提取相对来说耗时长、含有害化学物质、纯度低、RNA 完整度不高；而试剂盒提取的优点是效率高、纯度高、完整度高，各位可根据实际科研情况选择操作方法。

RNA 提取注意事项：
① 使用无 RNase 的塑料制品和枪头，玻璃器皿要消毒，避免交叉污染；使用性能较好的移液器，如 Eppendorf、Thermo 等；
② DEPC 有剧毒，小心配制；配制溶液应使用无 RNase 的水；
③ 操作人员应戴一次性口罩和手套，实验过程中要勤换手套；
④ 样品应避免反复冻融，否则影响提取 RNA 提取得率和质量；
⑤ 若下游实验对 DNA 非常敏感，可用不含 RNase 的 DNase I 对 RNA 进行处理。

2. 反转录注意事项
秉承避免 RNA 被污染和降解的原则，按照产品说明书进行即可，详细实验操作以及注意事项可参照之前已推出文章? 干货 | 实验篇之RT-PCR，此文对反转录过程的引物设计和注意事项给出了较明确的提示。

3. qPCR 引物设计注意事项
qPCR 过程中需要使用基因特异性引物扩增，从而分析目的基因表达量。
① 推荐使用软件 Primer Premier 5.0；
② qPCR 过程中使用引物长度推荐 25bp ，扩增产物长度 150bp ，可在 100bp-300bp 之间；
③ 正向引物和反向引物的 Tm 值相差不宜超过2℃，Tm 值在 60℃-65℃ 之间为佳；
④ 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；
⑤ 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；
⑥ 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；
⑦ 引物设计完成后需进行扩增效率检测，将扩增效率相同的引物用于定量比较分析。

4. 实验方法的选择
两步法：退火与延伸相同温度，最高可以达到72℃（很少采用），常用60℃，此温度下扩增引物二聚体和错配等扩增干扰较少，特异性高；每个循环时间较短，节约时间成本。
三步法：适合需要较高退火温度的引物，获得相对严谨的数据。

前期准备完毕，我们就可以进行接下来的实验了，为了避免踩到坑里，请仔细阅读！

二、qPCR 实验中期操作
中期操作主要分为试剂的存储、模板制备、体系配制、实验程序设置和内参选择几个部分，具体内容如下：

1. 试剂储存
-20℃ 避光长期储存，避免反复冻融，使用前充分混匀，但要避免产生气泡。部分试剂解冻后可能出现絮状物质，4℃ 放置并上下颠倒混匀至溶液澄清，不影响试剂性能。

2. 模板的制备
通常 1µg RNA 的反转录产物需要稀释10倍左右，以减轻 RNA 对 PCR 扩增的抑制。梯度稀释时，Ct 值落在15-28范围的稀释倍数作为原始模板稀释度，在此基础上进行5-10倍稀释。模板为 cDNA 原液时，上样量不超过 qPCR 反应总体积的1/10。

3. 体系配制
① 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。
② 推荐 10μL（q225、NOVO Max100）、20μL、50μL 体系；将引物和 qPCR Mix 混合配成大体系，混合均匀，为避免误差，可多配制1-2个加样孔的量，保证体系稳定性。
③ 通常引物终浓度为 0.2μM，也可以根据情况在 0.1-1.0μM 之间进行调整。

4. 实验程序设置
① 预变性推荐时间5min，根据不同模板和引物的具体情况可适当缩短至2min。此外，根据不同的厂家的产品可以有对应的预变性时间。
② 退火温度根据引物和目的基因的长度适当调整。
③ 仪器需定期校准，此处整理不同 ROX 适用机型，给各位小伙伴参考。

表1 不同荧光染料对应机型

5. 内参的选择
内参基因通常指各种管家基因，各类管家基因在生物体各类细胞中都表达。理想的内参基因可在各类细胞或组织、各种试验条件下恒定表达。但是不同物种、不同组织的管家基因表达存在特异性，目前还未发现完全理想的内参。下表是已报道的部分物种的 qPCR 内参基因，仅供参考。

表2 已报道部分物种的 qPCR 内参基因

如果表格中的数据无法满足您的需求，我们再支一招！

6. 如何筛选内参基因？
首先可以选择多个内参基因作为校正标准，再者可以使用 GeNorm、NormFinder 和 BestKeeper 比较内参基因表达的稳定性，使用软件 GeNorm、基因芯片数据和 EST 数据库筛选内参基因。

今天的知识点就这么多了，有没有拿小本本记好呢？

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1. 咨询当地客户经理；
2. 发送邮件至 enzyme@novogene.com；
3. 拨打文章底部客服咨询热线。
诺禾致源酶试剂业务线竭诚为您服务！

参考文献:
[1]董恩妮, 梁青, 李利, et al. 实时荧光定量PCR 内参基因的选择[J]. 中国畜牧杂志, 2013, 49(11):92-96.

酶试剂事业部 刘亭亭丨文案
孙津津丨编辑
图片来源于网络，侵删

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