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实验 | RNA提取实验中的那些玄学

发布: 2018-10-09 788阅读 218评论

当年还是新手的时候，曾经在网上读过不少关于提取RNA心得体会的文章。如今从事科研已经十年有余，现在回过头再去读那些文字，就会发现其中不少“经验”仅是“玄学”。这篇文章将破除一些迷信。

一、什么方案都比不过说明书

对于提取RNA的新手来说，无论是打算用试剂盒还是用Trizol法，首先需要阅读的是说明书，而不是网上的经验文（对，包括我这一篇）和实验方案。良心的试剂厂商在销售产品之前，会对其使用方案进行反复测试。因此，说明书中所提供的实验步骤，才是最准确的。

二、经典Trizol法并不能获得总RNA

这个事实可能会刷不少新手甚至老手的三观，但确有实验支持：经典Trizol法会选择性丢失低GC比的miRNA。这一点，源自一则作者自撤稿的故事。

V. Narry Kim是韩国鼎鼎大名的美女科学家，专做RNA相关的研究，包括miRNA。几年前她们组发现一个奇怪的“现象”，即贴壁细胞在消化之后，会有一些miRNA的丰度突然降低，看起来好像是被快速“降解”掉了，并据此写了一篇文章发到Molecular Cell上。但很快她们就发现，这个“现象”难以重复：如果用Trizol做实验，就一定是阳性结果，而用试剂盒提RNA，就重复不出来。难道是号称能提总RNA的Trizol方案出了问题？确实如此。

经进一步实验后发现，经典的Trizol方案会使得低GC比的miRNA丢失。如下图实验中，她们在不同总量或者说浓度的总RNA中，混入20 fmole的miR-141和miR-200c。然而，在总RNA量少的情况下，GC%比较低的miR-141，提取出来的相对浓度就会低于miR-200c。但这也不是说，Trizol法就完全不能用了。她们组还发现，在Trizol中加入10 mM的MgCl2，可以解决选择性丢失低GC比小RNA的问题。最后，V. Narry Kim写信给杂志社编辑部，要求撤除之前的文章。

试剂盒法，同样也是得看柱子是否支持抽提小RNA。目前市面上有仅富集小RNA、丢弃小RNA仅纯化大RNA，以及提取总RNA三种。总之又回到第一点，先看说明书，读完再动手。

三、对实验环境的要求并没有想象中那么苛刻

我在小本的时候曾去过某实验室实习，发现他们有专门的一个小隔离空间来提RNA，进去还得戴口罩。网上也有许多文章说，RNase很恐怖，所以到处都要擦得bling bling才能做实验。甚至还有许多试剂厂商推出了专门用来擦桌子的喷雾……对，RNase可怕这一点并不假，但也没有网上说得那么夸张，好像空气中弥漫的RNase都能榨出汁来的样子。

我本人提了那么多年RNA，从来就不用那些很夸张的措施。穿好实验服戴好手套，保护好自己就好。口罩爱戴不戴，反正我从来不戴。做实验时高冷一点，不要指点江山、口沫横飞即可。对了，勤换手套，甚至离一次心换一次的，也是玄学。

四、醇沉淀不是越多就越好

有些实验方案会推荐，在异丙醇沉淀后，用乙醇沉淀两次。实际上，任何提取实验，都会在纯度和得率这一对矛盾中取舍。醇沉淀会将脂溶性杂质去掉，但部分非脂溶性的杂质依然会连同RNA一起被沉淀下来。因此，多整几次，并不会让RNA的纯度翻倍，反而还会有降低得率的风险。一般情况下，异丙醇和乙醇各沉淀一次即可。但是，倘若预计RNA浓度很低，那就只能放弃纯度，在低温沉淀数小时，以换来较高的得率。

五、围绕DEPC的玄学

许多实验方案会推荐使用0.1% DEPC处理RNA相关用水，以灭活RNase。这一点是要肯定的。不过，在使用DEPC的过程中，又充斥着一些玄学。

首先，高压灭菌后的DEPC水，会有一股“香甜”的味道。许多人会以为那是残留的DEPC而不敢用。实际上，这只是DEPC分解成CO2和乙醇后，产生的一些挥发性酯类副产物。

其次，单纯高压过的蒸馏水或超纯水，基本可以视为无核酸酶活性等级的水。Thermo Fisher公司曾经做一项实验来说明这个问题。

（来源https://www.thermofisher.com/us/en/home/references/ambion-tech-support/nuclease-enzymes/tech-notes/rnase-and-depc-treatment.html）

如下图所示，在PBS中加入不同浓度RNase A，高压或不高压，后溶解P-32标记RNA 探针。最后数据显示，除非原始的RNase A的浓度高达1 µg/ml，否则常规高压过的水，基本检测不出RNase A活性。当然，Thermo的科学家也很谨慎，说这个结论仅适用于RNase A，不能推广到其他的RNase类型。

目前，我本人不使用DEPC来处理RNA实验相关试剂和耗材，因为太费劲，而且DEPC还有毒性。相关的液体试剂，我会使用从公司购买的Nuclease-free的水来配制。没有很贵，一小瓶够用很久。很多时候买各种其他试剂，比如酶、siRNA等，也都会送这样的水。在耗材方面，直接使用大公司的离心管和吸头，根本不用担心核酸酶的问题。

最后要注意的是，DEPC并不是万能的，尤其是无法处理含有Tris、HEPES等含胺类的溶液，除非加到很高的浓度。

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