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/A₂₈₀，A₂₆₀/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A_n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/A₂₈₀，A₂₆₀/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/A₂₈₀，A₂₆₀/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