Ctions focused on the pre-analytical phase [19]. The Horizon 2020 project SPIDIA4P aims to promote the standardization of pre-analytical workflows applied to customized medicine by developing European and international regular documents and external good quality assessment schemes [20]. Even so, to date, pre-analytical variability continues to be a critical situation whose implications wants further investigation. In this context, we made a study to assess the influence of pre-analytical variables, namely storage time and temperature among blood sampling and handling, on RNA from peripheral blood mononuclear cells (PBMCs). In distinct, we focused on evaluating alterations in RNA high-quality and gene expression occurring immediately after two h (h), 4 h, 6 h and 24 h delays in blood processing, at both 4 degrees Celsius ( ) and room temperature (RT). Our findings highlight the dramatic influence of pre-analytical processing on geneexpression and emphasize the relevance of correct blood sample handling and the require of share standardized procedures to get trusted results.MethodsSample collection and experimental designWhole blood samples were collected from 10 healthier subjects into 9 dipotassium ethylenediaminetetraaceticacid (K2-EDTA) VacutainerTM tubes (6 ml blood per tube, Becton ickinson, Franklin Lakes, NJ, USA). The time course study was performed as stick to: a single tube from every single participant was straight away processed for PBMC isolation (T0), the remaining tubes have been stored for two, 4, six or 24 h at either RT (i.e. 21 degrees Celsius) or 4 (Fig. 1). Two samples from 1 participant have been lacking, corresponding to six h storage at RT and 4 , as a result a total of 88 samples had been collected. Right after this phase, PBMCs had been isolated working with Lymphoprep (Stemcell Technologies) in accordance with typical procedures and stored at – 80 in RNA later (Invitrogen, Thermo Fisher Scientific) till RNA extraction. Sample collected were stored in CRESM Biobank, a structured biobank for neurological and autoimmune illnesses on the regional reference center for MS (CRESM), in Piedmont (Italy).Collagen alpha-1(VIII) chain/COL8A1 Protein supplier The study was approved by the Ethical committee of San Luigi Gonzaga University Hospital (approvals number 18390/2019).IL-2 Protein Gene ID Written informed consent was obtained from each participant.RNA extraction and high-quality controlTotal RNA was extracted utilizing the semi-automated Maxwell 16 Rapid Sample Concentrator (RSC) instrument with Maxwell R 16LEV basically RNA Tissue Kit (Promega, Madison, USA), following the manufacturer’s guidelines. Total RNAFig. 1 Graphical workflowMolecular Biology Reports (2022) 49:4709concentration was measured employing NanodropND-1000 spectrophotometer (Celbio, Italy). RNA purity was evaluated by examining the optical density (OD) ratios 260/280 and 260/230.PMID:24360118 RNA integrity was assessed making use of Bioanalyzer (Eukaryote Total RNA Nano kit, Agilent, Thermo Fisher Scientific) and RNA integrity numbers (RIN) have been calculated employing the 2100 Specialist software program (Thermo Fisher).Statistical analysisThe effect of storage time on RNA yield, top quality and integrity and on gene expression levels was examined at each and every temperature. Variations involving groups were evaluated by one-way repeated measures ANOVA followed by post hoc pairwise paired t-tests with Benjamini ochberg adjustment, or by Friedman’s test followed by Nemenyi’s post hoc test, as acceptable. Normality of distributions and sphericity assumption had been checked by Shapiro’s and Mauchly’s tests, respectively. Mean modifications within the Ct value of housekeeping genes had been ca.
