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

Abstract

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 10 4 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.

Non-Small Cell Lung Cancer Testing on Reference Specimens: An Italian Multicenter Experience

“…A secondary but equally limiting issue is the considerable heterogeneity among laboratories in managing the pre-analytical steps. This factor contributes to reducing even further the already scarce availability of diagnostic samples for the identification of DNA and RNA-based biomarkers [ 25 , 26 ]. In this scenario, harmonization of the analytical procedures is key to achieving timely and accurate analysis of clinically relevant biomarkers starting from very challenging diagnostic specimens [ 27 ].…”

“…As regards, purification of nucleic acids is another crucial factor to ensure accurate identification of predictive biomarkers [ 26 ]. Of note, we observed that a comparable number of institutions adopted manual and automated technical procedures for DNA and RNA purification.…”

“…In this scenario, harmonization of the analytical procedures is key to achieving timely and accurate analysis of clinically relevant biomarkers starting from very challenging diagnostic specimens [27]. In the present study, we strove to evaluate interlaboratory performance and reproducibility between 24 Italian laboratories by using a standard reference As regards, purification of nucleic acids is another crucial factor to ensure accurate identification of predictive biomarkers [26]. Of note, we observed that a comparable number of institutions adopted manual and automated technical procedures for DNA and RNA purification.…”

Comparison of transbronchial biopsy techniques using needle and forceps biopsies in lung cancer for molecular diagnostics: a prospective, randomized crossover trial

“…Nevertheless, the required tissue volume for NGS has not been determined and depends on the platform used ( 28 , 29 ). As genotyping is highly dependent on cytological preparation, fixation and staining techniques, there are no clear cut-off values, which may reduce reproducibility of these results ( 28 , 30 - 32 ). No additional slides were prepared for MVCA analysis, as we felt this may influence real world results.…”