Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
Arrakis Therapeutics, Waltham, MA, USA.
Methods Mol Biol. 2021;2252:127-149. doi: 10.1007/978-1-0716-1150-0_5.
Ribosome profiling is a genome-wide approach to map the positions of ribosomes on messenger RNAs. The abundance of ribosome-protected fragments can be used within condition to compare relative translation activities between different transcripts and between distinct conditions for the same transcript. A unified and routine method is currently lacking, however, to normalize between conditions for differences in global translation levels. Here we describe experimental and computational methods to use an orthogonal species spike-in, or internal standard, to enable absolute comparisons of translation activity between conditions. This simple modification of standard ribosome profiling provides a robust approach for accurately interpreting the effects of diverse genetic, chemical, and environmental perturbations of translation.
核糖体图谱分析是一种在全基因组范围内绘制核糖体在信使 RNA 上位置的方法。在条件下，核糖体保护片段的丰度可用于比较不同转录本之间以及同一转录本在不同条件下的相对翻译活性。然而，目前缺乏一种统一的常规方法来对不同条件下的整体翻译水平差异进行标准化。在这里，我们描述了使用正交物种掺入物或内部标准的实验和计算方法，以实现条件之间翻译活性的绝对比较。这种对标准核糖体图谱分析的简单修改为准确解释遗传、化学和环境对翻译的各种扰动的影响提供了一种可靠的方法。
