Optimization of pre‐analytical and analytical steps for DNA and RNA analysis of fresh cytology samples

• Published in: Cancer medicine (Malden, MA) MA), 2022-11, Vol.11 (21), p.4021-4032
• Main Authors: Dolinar, Ana, Grubelnik, Gašper, Srebotnik‐Kirbiš, Irena, Strojan Fležar, Margareta, Žlajpah, Margareta
• Format: Article
• Language: English
• Subjects: Acids; automated extraction; Automation; Cell lines; Cellular biology; cytological samples; Cytology; Deoxyribonucleic acid; DNA; DNA - analysis; DNA - genetics; Ethanol; Experiments; extraction methods; extraction with TRI reagent; Humans; Laboratories; Molecular Diagnostic Techniques; Mutation; Nucleic Acids - analysis; Reagents; Ribonucleic acid; RNA; RNA - genetics; spin‐column extraction; Storage conditions
• ISSN: 2045-7634; 2045-7634
• DOI: 10.1002/cam4.4728

Background 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. Methods 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. Results 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 104 cells, while specific mutation was detected in as low as 5% of mutated cells. Conclusions 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. Using cell line model to mimic fresh cytology samples, we have determined the influence of storage conditions (time, temperature) on the quality and quantity of nucleic acids for cells suspended in a buffer‐based cell medium. Additionally, we assessed the minimal number of cells and minimal percentage of mutated cells per sample for successful molecular testing.