迈出脚步
体验世界

RNA浓度测定中A260/A280,A260/A230到底是什么?有什么意义?偏高偏低怎么办?

在提取RNA并测定RNA浓度的时候,非常容易看到的两个概念是A260/A280A260/A230,那么这两个比值到底能说明什么?

根据朗博-比尔定律,化学物质可吸收一定波长的光,且对光的吸收度A与此化学物质的浓度c成正比,因此可用吸光度大小来测定化学物质的浓度,

A=lg(1/T)=Kbc

某物质在某一特定波长n下吸光度值可用An来表示

  • A280,因为蛋白质含有芳香氨基酸,因此也能吸收紫外光,且吸收峰在280nm波长处,因此A280可代表蛋白质浓度
  • A260,这个就是RNA的吸收峰,可反应RNA浓度值
  • A230,在提取RNA过程中所用到的试剂,裂解液、结合液、漂洗液、洗涤液和洗脱液中会有TRIzol、硫氰酸胍、盐酸胍等离液盐、SDS、Triton X-100、Tween 20等去污剂、苯酚、EDTA、乙醇、异丙醇、NaCl等物质。尤其是硫氰酸胍,都可能能会导致A230较高,同时降低A260/A230的比值。

    关于A260/A230这个比值的影响因素以及重要性,其实没有完全的定论,Qiagen公司说到由硫氰酸胍引起的A260/A230 比值偏低对后续实验影响并不大,即使当硫氰酸胍浓度高达100mM时,也同样如此

    However, we also found that concentrations of guanidine thiocyanate of up to 100 mM in an RNA sample do not compromise the reliability of real-time RT-PCR, even when using PCR chemistries that are sensitive to inhibitors

    点此下载Qiagen原文件说明

 

普遍认可的是A260/A280在1.8~2.1之间比较合适,A260/A230越接近2越好。但其实,如果完全撇开浓度去谈这两个比值,都是在刷流氓

美国NEB做过这样一个测试,以Thermo生产的 NanoDrop© One 进行测定A260/A280A260/A230的准确性

a dilution series of 150 ng/µl down to 1 ng/µl in TE buffer was analyzed. Measurements were carried out in triplicate and data were collected for concentration and purity ratios

最终得出的结论是:当浓度低于50ng/uL时,A260/A280A260/A230的比值波动非常大,没有太大的意义,当浓度高于50ng/uL时,比值波动比较小,可以信赖

A260/A280 ratios are unusable at concentrations <20 ng/µl , as indicated by the variability in triplicate measurements. Remarkably, the variability is still relatively high in the 20–50 ng/µl range, with averages tending to be too high. Only when working with higher concentrations, can consistent and reliable values be obtained.

A260/A230 ratios show a similar trend. They are unusable at concentrations below 20 ng/µl and should be used with care between 20–50 ng/µl: the A260/A230 ratio is too high and shows significant variability. Above 50 ng/µl the values are more reliable. Overall, A260/A230 ratios have a higher standard deviation than A260/A280 ratios. Similar trends were observed with RNA.

点此下载NEB原文

http://xzh.i3geek.com
赞(0) 喜欢我
未经允许不得转载:王威 » RNA浓度测定中A260/A280,A260/A230到底是什么?有什么意义?偏高偏低怎么办?
分享到: 更多 (0)

评论 抢沙发

  • 昵称 (必填)
  • 邮箱 (必填)
  • 网址

觉得文章有用就打赏一下文章作者

支付宝扫一扫打赏

微信扫一扫打赏