Elettra-Sincrotrone Trieste, S.S. 14 Km 163.5, 34151, Trieste, Italy.
Dipartimento di Fisica, Università degli Studi di Trieste , via Valerio 2, 34127 Trieste, Italy.
Anal Chem. 2016 Dec 20;88(24):12090-12098. doi: 10.1021/acs.analchem.6b02744. Epub 2016 Nov 30.
We report on an optimized protocol for the digestion of cellular RNA, which minimally affects the cell membrane integrity, maintaining substantially unaltered the vibrational contributions of the other cellular macromolecules. The design of this protocol allowed us to collect the first Fourier transform infrared (FTIR) spectra of intact hydrated B16 mouse melanoma cells deprived of RNA and to highlight the in-cell diagnostic spectral features of it. Complementing the cellular results with the FTIR analysis of extracted RNA, ds-DNA, ss-cDNA and isolated nuclei, we verified that the spectral component centered at ∼1220 cm is a good qualitative and semiquantitative marker of cellular DNA, since it is minimally affected by cellular RNA removal. Conversely, the band centered at ∼1240 cm, conventionally attributed to RNA, is only a qualitative marker of it, since its intensity is majorly influenced by other macromolecules containing diverse phosphate groups, such as phospholipids and phosphorylated proteins. On the other hand, we proved that the spectral contribution centered at ∼1120 cm is the most reliable indicator of variations in cellular RNA levels, that better correlates with cellular metabolic activity. The achievement of these results have been made possible also by the implementation of new methods for baseline correction and automated peak fitting, presented in this paper.
我们报告了一种优化的细胞 RNA 消化方案，该方案对细胞膜完整性的影响最小，同时保持了其他细胞大分子的振动贡献基本不变。该方案的设计使我们能够收集第一个傅里叶变换红外（FTIR）光谱的无 RNA 的完整水合 B16 小鼠黑色素瘤细胞，并突出其细胞内诊断光谱特征。通过对提取的 RNA、ds-DNA、ss-cDNA 和分离的核进行 FTIR 分析，补充细胞结果，我们验证了位于约 1220 cm 的光谱分量是细胞 DNA 的良好定性和半定量标记物，因为它受细胞 RNA 去除的影响最小。相反，位于约 1240 cm 的谱带，通常归因于 RNA，只是其定性标记物，因为其强度主要受到其他含有不同磷酸基团的大分子的影响，如磷脂和磷酸化蛋白。另一方面，我们证明了位于约 1120 cm 的光谱贡献是细胞 RNA 水平变化的最可靠指标，与细胞代谢活性的相关性更好。本文还介绍了新的基线校正和自动峰拟合方法的实施，这些方法使得这些结果的实现成为可能。
