前面给大家着重介绍了 qPCR 的实验流程和注意事项【干货 | 实验篇之荧光定量 PCR（上）】想必大家意犹未尽，今天小编又双叒叕带来荧光定量实验下篇，主要为大家讲解 qPCR 实验后的结果分析与数据处理。
首先给大家讲解一下 qPCR 的结果分析，最主要的就是实验中经常出现的三大曲线：扩增曲线、溶解曲线、标准曲线。
图1
1. 扩增曲线：扩增曲线有两种展现形式，一种是线性，一种是对数形式。我们通常是用 Ct 来推算样品中样品的浓度。Ct 值越高说明模板浓度越低。
2. 熔解曲线：Tm 值，Melting Temperature（解链温度），是 PCR 双链产物的退火温度。在达到其解链温度时，荧光信号会有一个忽然的下降，将测得的这个曲线叫做熔解曲线。理论上如果 PCR 得到特异性产物则只有一个 Tm 值，在溶解曲线上表示只有单峰存在。如果是多相峰，那么可以判断产物不是单一的，发生了非特异扩增。
3. 标准曲线： 将已知浓度的样品（标准品）经过梯度稀释后分别取样进行荧光定量 PCR，得到的一系列的 Ct 值，用这个 Ct 值与 Log 模板数对应可以得到一个相关的曲线，我们叫标准曲线。可以用这个标准曲线中的一些参数来判断这个荧光定量 PCR 体系的优劣。
qPCR 中的“奇葩结果”及“对症下药”之法
| 扩增曲线：
| 熔解曲线：
| 标准曲线：
数据分析
在 qPCR 中，一般分为绝对定量和相对定量：
1. 绝对定量中，起始浓度与循环数呈线性关系。可通过已知起始拷贝数的标准品制作出标准曲线，得到该扩增反应存在的线性关系。然后根据样品的 Ct 值，来确定未知样品的初始模板拷贝数或浓度。通常用于病毒、细菌、衣原体、支原体的定量检测及转基因食品的检测。
2. 相对定量则用于测定单个样品基因的差异表达分析或者比较两个或两个以上样品中某个基因表达量的变化，则其结果必须用内参基因（管家基因）校正。
相对定量的分析方法主要有两种：双标准曲线法和 2^-△△Ct 法。
双标准曲线法： 每次实验都要分别用标准品做内参基因和目的基因的标准曲线，并同时扩增各待测样本中的目的基因和内参基因。然后使用标准曲线来计算待测样本中的目的基因和内参基因的表达量。最后通过公式就可以计算出目的基因的表达差异了。
2^-△△Ct 法： 是最常用的方法，其得到的结果是实验组中目的基因相对于对照组中目的基因表达的差异倍数。要求目的基因和内参基因的扩增效率相同，且相对偏差不超过5%。其原理是：在平台期的所有基因扩增的数目是一致的。而唯一有区别的则是 Ct 值的不同。Ct 值是每个反应管内的荧光信号达到设定的域值时所经历的循环数(cycle)。所以不难推断出 Ct 值越小，反应扩增到达平台期所需循环数越少，目的基因起始含量越高。
下面以细胞内 p53mRNA 和 GAPDH 为例利用 2^-△△Ct 法为大家讲解如何处理实际的数据。
图1
其中 A 组为处理后的样本，而 B 组为对照组，每个样本重复三次。一般来说，我们最好将同一组织同一基因进行多次独立实验重复，就是说我们要在其他时间多做几次实验，提 RNA，再逆转录，再做 qPCR，如此做了三次实验后才能算作独立实验重复，我们统计的时候统计的是独立实验重复，这样能最大的保证实验的精确度和正确性。
做完 qPCR 实验后，我们得到以下结果（均为 Ct 值）：
表1
结果计算与分析：
第一步，计算各次独立实验平均 Ct 值和 △Ct 值：其中 △Ct=Ct(p53)-Ct(GAPDH)，
第二步，计算每组每次独立实验的 2^-△Ct 值，
第三步，计算 2^-△△Ct 值，其中 2^-△△Ct=所有 2^-△Ct 值/B组 2^-△Ct 的平均值，
表2
第四步，对 2^-△△Ct 求平均值和标准差：
表3
虽然均值和标准差都有了，虽然看上去相差那么大，如何确定 P 值呢？可以采用 SPSS 统计软件来统计 P 值。需要强调的是，因为都是独立的样本，所以采用独立样本t检验方法来检验。
今天简单给大家介绍了一下基本的 qPCR 结果分析，在实际的实验过程中，不同的实验设计都会有各种各样的分析方法，还需要各位小伙伴根据自己的实验进行调整。与此同时，诺禾致源专为 qPCR 而生的一项爆款产品，让你的 qPCR 完美无缺！
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