Analysis of RNA quantitation in formaldehyde-fixed cells

Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize cellular subpopulations and great advances have been made using this approach. Cells are often fixed with formaldehyde prior to the sorting process to preserve cell morphology and maintain the expression of surface molecules, as well as to ensure safety in the sorting of infected cells. It is widely recognized that formaldehyde fixation alters RNA and DNA structure and integrity, thus analyzing gene expression in these cells has been difficult. We therefore examined the effects of formaldehyde fixation on the stability and quantitation of nucleic acids in cell lines, primary leukocytes and also cells isolated from SIV-infected pigtailed macaques. We developed a method to extract RNA from fixed cells that yielded the same amount of RNA as our common method of RNA isolation from fresh cells. Quantitation of RNA by RT-qPCR in fixed cells was not always comparable with that in unfixed cells. In comparison, when RNA was measured by the probe-based NanoString system, there was no significant difference in RNA quantitation. In addition, we demonstrated that quantitation of proviral DNA in fixed cells by qPCR is comparable to that in unfixed cells when normalized by a single-copy cellular gene. These results provide a systematic procedure to quantitate gene expression in cells that have been fixed with formaldehyde and sorted by FACS. We compared RNA quantitation between unfixed and formaldehyde-fixed cells using the NanoString nCounter system. This analysis was performed using two cell lines, K562 and CEMx174, and human PBMCs. Three biological replicates were performed for each cell type. We also performed this comparison using human PBMCs sorted by FACS. For this analysis, we isolated PBMCs from two donors and performed two technical replicates for each donor sample.

荧光激活细胞分选术（Fluorescence-activated cell sorting, FACS）是一种灵敏且极具应用价值的细胞亚群表征技术，依托该技术已取得诸多重要研究进展。在分选流程启动前，通常会使用甲醛对细胞进行固定，以维持细胞形态、保留表面分子的表达水平，并确保感染细胞分选过程的生物安全性。
学界普遍认为，甲醛固定会改变核糖核酸（RNA）与脱氧核糖核酸（DNA）的结构与完整性，因此对此类细胞开展基因表达分析曾一度存在较大难度。
为此，本研究针对甲醛固定对细胞系、原代白细胞以及从猴免疫缺陷病毒（Simian Immunodeficiency Virus, SIV）感染的猪尾猕猴体内分离得到的细胞中核酸的稳定性与定量分析的影响展开了系统性探究。
我们开发了一种从固定细胞中提取RNA的方法，其RNA得率与常规从新鲜细胞中分离RNA的方法相当。通过逆转录实时定量聚合酶链反应（RT-qPCR）对固定细胞中的RNA进行定量时，所得结果未必能与未固定细胞的定量结果保持一致。相较而言，若采用基于探针的NanoString nCounter系统进行RNA定量，则未观察到两组间存在显著差异。
此外，本研究证实，当以单拷贝细胞基因作为内参进行归一化处理后，通过定量聚合酶链反应（qPCR）对固定细胞中的前病毒DNA进行定量的结果，与未固定细胞的定量结果具有可比性。本研究所得结果为经甲醛固定并通过FACS分选的细胞的基因表达定量分析提供了一套标准化操作流程。
我们采用NanoString nCounter系统对比了未固定细胞与甲醛固定细胞的RNA定量结果，该分析涵盖了K562、CEMx174两种细胞系以及人外周血单个核细胞（Peripheral Blood Mononuclear Cells, PBMCs）。每种细胞类型均设置了三次生物学重复。
我们还利用经FACS分选的人外周血单个核细胞开展了上述对比分析：从2名供者体内分离PBMCs，并对每份供者样本设置两次技术重复。