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However, it is known to be vulnerable to pre-analytical variables. Although RNA stabilization systems have been shown to reduce such influence, traditional EDTA tubes are still widely used since they are less expensive and enable to study specific leukocyte populations. This study aimed to assess the influence of storage time and temperature between blood sampling and handling on RNA from peripheral blood mononuclear cells (PBMCs).<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Methods and results<\/jats:title>\n                <jats:p>Nine blood samples were collected in EDTA tubes from 10 healthy donors. One tube from each donor was immediately processed for PBMC isolation, while the others were first incubated at either 4 degrees Celsius (\u00b0C) or room temperature for 2, 4, 6 and 24\u00a0h. RNA yield and quality and the expression level of fourt housekeeping (B2M, CASC3, GAPDH, HPRT1) and 8 target genes (CD14, CD19, CD20, IL10, MxA, TNF, TNFAIP3, NR4A2) were compared between samples.<\/jats:p>\n                <jats:p>RNA yield, quality and integrity did not vary significantly with time and temperature. B2M was the most stable housekeeping gene, while the others were increasingly influenced by storing time, especially at 4\u00a0\u00b0C. Even when normalized to B2M, the expression level of some target genes, particularly TNFAIP3 and NR4A2, was highly affected by delays in blood processing at either temperature, already from 2\u00a0h.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusion<\/jats:title>\n                <jats:p>Pre-analytical processing has a great impact on transcript expression from blood collected in EDTA tubes, especially on genes related to inflammation. Standardized procedure of blood handling are needed to obtain reliable results.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1007\/s11033-022-07320-5","type":"journal-article","created":{"date-parts":[[2022,3,12]],"date-time":"2022-03-12T17:02:41Z","timestamp":1647104561000},"page":"4709-4718","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["The impact of pre-freezing storage time and temperature on gene expression of blood collected in EDTA tubes"],"prefix":"10.1007","volume":"49","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6889-888X","authenticated-orcid":false,"given":"Serena","family":"Martire","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paola","family":"Valentino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fabiana","family":"Marnetto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luca","family":"Mirabile","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marco","family":"Capobianco","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Antonio","family":"Bertolotto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,3,12]]},"reference":[{"key":"7320_CR1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0112293","author":"F Malentacchi","year":"2014","unstructured":"Malentacchi F, Pazzagli M, Simi L et al (2014) Spidia-RNA: second external quality assessment for the pre-analytical phase of blood samples used for rna based analyses. 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VP received speaker honoraria from Biogen and Novartis, research support from Merck and grant support from Quanterix. MF received speaker honoraria from Biogen and research support from Sanofi Genzyme. ML has nothing to disclose. CM served on the scientific advisory board of Biogen, Sanofi Genzyme, Novartis, and Bayer Schering and received speaker honoraria from Almirall, Biogen, Novartis, Sanofi Genzyme, and Teva. BA served on the scientific advisory board of Almirall, Bayer, Biogen, and Genzyme; received speaker honoraria from Biogen, Novartis and Sanofi and grant support from Almiral, Biogen, Associazione San Luigi Gonzaga ONLUS, Fondazione per la Ricerca Biomedica ONLUS, Mylan, Novartis and the Italian Multiple sclerosis Society.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Conflict of interest"}},{"value":"The study was approved by the Ethical committee of San Luigi Gonzaga University Hospital (approvals number 18390\/2019). Written informed consent was obtained from each participant.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethical approval"}}]}}