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一文教你如何从细胞中提取高质量RNA

发布: 2017-05-26 897阅读 203评论

在实验室中，尤其是分子生物类的实验室中不可避免的会遇到细胞总RNA提取实验，所提RNA样品的质量和纯度直接影响下一步的实验，如逆转录，RT-PCR, microarray 等。下面我们来介绍如何提取细胞中的总RNA 。

首先，我们要知道什么是总RNA。总RNA包括mRNA和miRNA，其中mRNA也就是信使RNA，是具有编码蛋白质的功能的RNA，传递遗传信息。miRNA是小型非编码RNA，长度约22个碱基，可以调控或沉默基因表达编码RNA。我们的目的是得到质量好，纯度高，完整的RNA样品。操作如下：

细胞裂解
细胞的获取方式不同，采用的裂解方法也不尽相同。

1）若是组织中提取，按照每50-100mg样品加入1mlTrizol的比例加入Trizol，而后匀浆破碎样品。细胞破碎后在室温下静置5分钟，这样可以分离出核蛋白复合物（nucleoprotein complexes），然后离心去除细胞碎片[2]。
2）若是培养瓶中贴壁细胞，则先用冰PBS洗2次，而后按每10平方厘米区域加入1ml Trizol的比例加入Trizol, 吹打混匀处理。
3）若是悬浮细胞，离心收集细胞，去除培养基，然后加入适量Trizol吹打混匀处理。

可以发现这步操作主要用到了Trizol这个试剂。那么Trizol的作用是什么呢？Trizol的主要成分是苯酚，是有效的蛋白变性剂。它能够抑制酶活性包括RNAaes，这样可以保证RNA不会被降解掉，同时Trizol能够使细胞裂解。

抽提RNA
向EP管中的细胞液加入200ul氯仿，涡旋15秒后，室温静置3分钟，然后4℃，12000rpm,15min条件下离心。离心后分3层，上层为无色或淡粉色含有RNA，中层为白色蛋白质层，下层为红色含有脂质和培养基。我们所需的RNA就在氯仿的作用在分在上层的水相中。

氯仿是有机溶剂，添加氯仿会使细胞内的蛋白变性沉淀，可以通过离心进行去除。同时加入氯仿后，水相和有机相会分层，将水相中的酚抽提以免损伤核酸，另外还能溶解一些脂溶性杂质纯化RNA。

沉淀RNA
吸取上层水相于新的EP管中，不要吸取中间蛋白质层，加入等体积的异丙醇，一般是500ul上清和500ul异丙醇，少的时候可以取200ul的上清。两者充分混匀，室温放置10分钟,然后4℃，12000rpm离心10分钟。离心结束后，弃去上清，管底可见白色胶状沉淀，这就是我们的RNA了。

好奇心强的朋友一定会问了，加入异丙醇的作用是什么呢？异丙醇的作用就是沉淀水相中的RNA并抑制RNA酶活性，其羟基的疏水作用能保护RNA中的亲水基团。

洗涤RNA
为了进行RNA的洗涤，我们需要用DEPC水配制75%的乙醇溶液，现配现用。哪尼？你不知道什么是DEPC水。DEPC水呢，就是是用DEPC(diethyl pyrocarbonate，焦碳酸二乙酯)处理过并经高温高压灭菌的MiliQ纯水，里面不含杂质RNA，DNA和蛋白质。

按照添加的Trizon的量1:1体积配乙醇溶液，一般是750ul分析纯乙醇加上250ul DEPC水，在涡旋仪上使乙醇溶液与RNA充分混匀，4℃，9000rpm,5min离心。然后小心弃去上清，不要吸走管底的RNA哦。

再次洗涤RNA
重复第四步骤，再次洗涤RNA，弃去上清后，控干EP管。，室温下将EP管倒置在吸水纸上5-10min,酌情加入20-50ul DEPC水溶解RNA，这就是我们最终提取到的RNA了。

测定RNA纯度和浓度
用微量紫外分光光度计取1-2ul RNA检测即可。A260/A280值在1.8-2.0间符合纯度。RNA在260nm处有最大吸收，A260=1.0代表RNA的浓度为40ug/ml。A280用于检测蛋白污染，纯RNA样品的A260/A280=2.1，一般所得比值在1.8-2.0间也是普遍认可的。另外，我们也可以测定A230。A230用于检测其他污染，主要来自RNA提取试剂，理想的A230值要大于1.5。另外RNA的完整性可以用1%的琼脂糖凝胶电泳验证[2,3]。

操作注意事项：
1. 在收集细胞时，离心操作过程中离心管中看不见明显的细胞沉淀时，可以在倒置显微镜下观察培养瓶细胞残留，判断细胞是否都消化下来。另外，可以适当延长离心时间。

2.加入氯仿时，要剧烈混匀使两相彻底混匀 。DNA在水饱和酚的酸性条件下是会被留在有机相的，不会跑水相里头去。吸取上清液时尽量不要分到不同的新的EP管中，本身量不多时，尽量集中到一个管中，同时也避免吸到中间蛋白降低纯度。

3.加入异丙醇后没有看到明显的乳白色沉淀时，可以在-20℃过夜再离心，也可以继续按照实验操作往下做。

每个实验室的提取方法可能不尽相同，此处的方法仅供参考交流。在实验过程中遵守操作规范，严谨实验，这样便能大大提高实验结果的准确性和可靠性。

参考文献：
[1] Chi, K. R. The RNA code comes into focus. Nature 542, 503-506, doi:10.1038/542503a (2017).（封面图来源）
[2] http://bitesizebio.com/13529/isolation-of-total-rna-including-microrna-from-cells-and-tissues/
[3] http://bitesizebio.com/13527/how-to-quality-control-check-your-rna-samples/

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