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干货 | 实验篇之荧光定量 PCR(上)

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?

想了解更多,请联系我们:
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

作者: 诺禾科服

国内规模领先的高通量测序公司,高通量测序,生物信息分析,科研服务,临床检测。微信公众号:novogene-1



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