Department of molecular genetics, Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
Department of cytopathology, Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
Cancer Med. 2022 Nov;11(21):4021-4032. doi: 10.1002/cam4.4728. Epub 2022 Apr 10.
Different cytology preparations can be used for molecular diagnostics, however the influence of pre-analytical and analytical steps on the results are not yet well defined. We aimed to determine optimal steps for efficient extraction of DNA and RNA from fresh cells for molecular diagnostics.
MCF7 and FaDu human cell lines, were used as a model to determine fresh cells storage conditions (temperature: 25°C, 4°C, -20°C, -80°C; duration: 0 h, 4 h, 12 h, 24 h, 48 h) and optimal nucleic acids extraction method. Besides, the minimal number of total cells and minimal percentage of mutated cells needed for successful extraction of nucleic acids and subsequent determination of present mutation were evaluated.
Extraction of nucleic acids using spin columns yielded the highest quantity and quality of nucleic acids. Isolation of nucleic acids was feasible in all storage conditions, however higher temperature and longer duration of fresh cells storage were associated with lower quality of isolated nucleic acids and similar quantification cycle of housekeeping genes. Successful molecular testing was feasible with least 10 cells, while specific mutation was detected in as low as 5% of mutated cells.
Our cell line model, mimicking fresh cytology samples, showed that quantity of extracted either DNA or RNA declined with higher temperatures and longer duration of storage but regardless of the storage conditions, we successfully detected both housekeeping genes and mutated gene using qPCR.
不同的细胞学标本可用于分子诊断，但分析前和分析步骤对结果的影响尚不清楚。我们旨在确定从新鲜细胞中高效提取 DNA 和 RNA 以用于分子诊断的最佳步骤。
MCF7 和 FaDu 人细胞系被用作模型，以确定新鲜细胞的储存条件（温度：25°C、4°C、-20°C、-80°C；持续时间：0 h、4 h、12 h、24 h、48 h）和最佳核酸提取方法。此外，还评估了成功提取核酸和随后确定存在突变所需的总细胞数最少和突变细胞百分比最小。
使用离心柱提取核酸可获得最高数量和质量的核酸。在所有储存条件下都可以分离核酸，但较高的温度和较长的新鲜细胞储存时间与分离核酸的质量较低以及管家基因的定量循环相似相关。至少需要 10 个细胞即可进行成功的分子检测，而在低至 5%的突变细胞中即可检测到特定的突变。
我们的细胞系模型模拟新鲜细胞学标本，结果表明，无论储存条件如何，提取的 DNA 或 RNA 的数量均随温度升高和储存时间延长而减少，但我们使用 qPCR 成功检测了管家基因和突变基因。
