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干货 | 实验篇之RT-PCR

发布: 2018-12-22 1,147阅读 207评论

作者：高天政

逆转录实验作为是常见的分子生物学实验之一，虽然看上去步骤简单，但当你实际操作时就会发现各种问题扑面而来，相信各位小伙伴都有被一个简单的逆转录实验搞得头晕眼花的时候，今天就由小编给大家介绍一下逆转录实验中的注意事项和常见问题，希望大家学以致用哦！

图1. 逆转录过程

逆转录（reverse transcription）是以 RNA 为模板合成 DNA 的过程，即 RNA 指导下的 DNA 合成，逆转录实验包括3大要素，分别是引物、逆转录酶和 RNA 模板：

1. 引物：逆转录引物主要有3种，包括 Oligo（dT）、Random Primers 和基因特异性引物（GSP）。其中 Oligo（dT）具有12-20个 T 碱基，并且与真核生物 mRNA 的 3’Poly A 尾配对，可合成全长的 cDNA，但仅扩增有 polyA 尾的 mRNA ，对模板质量要求高，较适合克隆实验。而 Random Primers 具有6-9个碱基，可随机识别模板并结合，适合复杂结构和微量模板，但特异性低，小片段多，适合后续 qPCR 实验。基因特异性引物可以识别特定模板序列，具有特异性强，灵敏度高的特点，但需合成特定的序列。所以根据不同实验需求可进行相应引物的选择。

2. 逆转录酶：一种是来自纯化的禽成髓细胞瘤病毒（AMV），由两条肽链组成，具有聚合酶活性和很强的 RNaseH 活性，它最适温度是42℃，最适 pH8.3，在高反应温度时可消除 mRNA 的二级结构对逆转录的阻碍，然而高水平的 RNaseH 的活性既抑制 cDNA 产生也限制其长度。另一种来源于鼠白血病病毒（M-MLV），是单肽链的，有 RNA 聚合酶活性和相对较弱的 RNaseH 活性，最适温度37℃，最适 pH7.6，较弱的 RNaseH 活性对获得2-3kb的 mRNA 的全长 cDNA 有很大好处。

3. RNA 模板：通常利用紫外分光光度计检测纯度，要求A260/280=1.9~2.1，A260/230=2.0~2.2。利用琼脂糖凝胶电泳检测 RNA 的完整度，完整的总 RNA 在进行琼脂糖凝胶电泳时会产生清晰的28S 和18S rRNA 条带（真核样品），28S rRNA 条带的强度应当大致为18S rRNA 条带的两倍，并且无 gDNA 和蛋白残留。

图2. 琼脂糖凝胶电泳检测 RNA 完整性

上面给大家简单介绍了一下逆转录的3大要素，除此之外在实际的操作过程中，还会有各种各样的让问题我们措手不及，下面给大家一一详细解答！

1. 如何提高 RT-PCR 反应的灵敏度与特异性？

① 确定模板 RNA 完整性好，无 DNA 污染。
② RNA 模板中不应含有扩增反应抑制剂。
③ 使用适量的模板 RNA ，模板量太多会降低特异性，太少会导致扩增不出条带或条带太弱。
④ 若模板中有二级结构，可通过提高逆转录反应温度来提高扩增效果。

2. RNA 中含有逆转录抑制剂时，怎么处理？

逆转录抑制剂包括：SDS 、EDTA 、甘油、焦磷酸钠、亚精胺和胍盐等等。可将已确定的高质量 RNA 模板同样品混合，同高质量 RNA 模板比较产量以检测 RNA 抑制剂；若高质量模板 RNA 与样品混合后产量降低，则说明样品中存在逆转录抑制剂，可用70%（v/v）乙醇对 RNA 沉淀进行清洗，以除去抑制剂。

3. 逆转录后的 qPCR 实验 Ct 值偏大或者普通 PCR 产量低。

① RNA 模板质量差，需要重新制备 RNA 模板，可通过琼脂糖凝胶电泳检测质量。
② 逆转录后的 cDNA 中含有高浓度的模板 RNA 和逆转录试剂成分，可能会对后续的PCR 产生抑制作用，所以可以适当梯度提高 cDNA 稀释比例（通常来说可将 cDNA 原液稀释5-10倍，其最佳模板加入量以扩增得到的 Ct 值在20-30个循环为好）。
③ 起始的 RNA 模板量低，需要减少稀释倍数或增加 RNA 模板量。
④ 基因本身原因（复杂和长度），可以重新设计复杂基因的引物，避免复杂结构。或者用三步法程序或延长两步法程序的延伸时间。
⑤ 逆转录或者定量产品性能差，可以通过做平行不用品牌产品对比实验，对比逆转录产品性能。

4. 逆转录酶扩增效率评估，应该关注哪些指标？

建议： qPCR-ct 值，或者 PCR 产量
如果想比较逆转录性能，最为直接的还是后续做 qPCR 或者 PCR 平行对比实验，这种方法相对较为准确。
不建议：cDNA 中间产物浓度测定 or 其他方法。
逆转录完成后是不建议测定产物浓度的，由于逆转录后产生 RNA-cDNA 杂交链，溶液中的 RNA 和部分 DNA 会对吸光值产生影响，所以测定出来的产物浓度并不准确，即使利用同一批次 RNA 和不同品牌的试剂盒进行对比实验，由于不同品牌之间的逆转录体系具有或多或少的差异，其体系中的各种离子等都能对吸光度产生影响，所以测定的结果也没有可比性。

优化及注意事项
① 逆转录 PCR 时，提取 RNA 是关键，需分离高质量 RNA（纯度和完整性）。
② 整个操作过程需使用去 RNA 酶的枪头、EP 管等耗材。
③ 逆转录过程中要谨防 RNA 酶的污染，加入 RNA 酶抑制剂。
④ 为了防止非特异性扩增，必须设阴性对照。
⑤ 逆转录实验应全程在冰上操作完成，操作过程应避免 RNase 污染。
⑥ 提高逆转录预热温度（也可根据相应逆转录试剂盒进行调整）。
⑦ 减少基因组 DNA 污染，可使用去基因组的逆转录试剂盒。

Tip

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