Preanalytical Procedures for DNA Studies: The Experience of the Interinstitutional Multidisciplinary BioBank (BioBIM)

Abstract

Preanalytical variables, including the anticoagulants and stabilizing agents, time, storage temperature, and methods of DNA extraction applied to blood samples, may affect quality and quantity of isolated nucleic acids for future genomic applications. Considering the large number of collected samples, standard operating procedure optimization for whole blood preservation before DNA extraction is a crucial step in a biological repository. Moreover, the future application of the biological material may not be known subsequent to its extraction. To define standard operating procedures for whole blood preservation before DNA extraction, we aimed to determine whether different combinations of anticoagulants, blood storage temperatures, and time intervals before storage at -80°C might have an impact on quality and quantity of subsequent extracted DNA. After spectrophotometer quantification, the quality and integrity of DNA were assessed by agarose gel electrophoresis, polymerase chain reaction, and real-time polymerase chain reaction methods. We observed that decrease in DNA recovery during blood storage time was more pronounced at room temperature than at 4°C. Based on our experience, we recommend as anticoagulants of choice sodium citrate and ethylenediaminetetraacetate, whereas sodium citrate theophylline adenosine dipyridamole could represent an alternative choice, excluding a priori lithium heparin and Fluoride-Oxalate. Based on the overall evaluation criteria, we conclude that the procedures necessary to preserve the whole blood before the DNA extraction may have a significant impact on downstream molecular biological applications.

Role of genetic polymorphisms of ion channels in the pathophysiology of coronary microvascular dysfunction and ischemic heart disease

“…In conformity with the study protocol, ethylenediaminetetraacetic acid (EDTA) whole blood samples were collected according to the international guidelines reported in the literature [48]. Samples were transferred to the Interinstitutional Multidisciplinary BioBank (BioBIM) of IRCCS San Raffaele Pisana (Rome) and stored at −80 °C until DNA extraction.…”

Influence of pre-analytical procedures on genomic DNA integrity in blood samples: The SPIDIA experience

“…Pre-analytical variables, including storage temperature and time and DNA extraction methods, may affect the quality of isolated DNA for molecular application [2] and thus affecting analytical results [26]. An important part of the SPIDIA project, a four-year integrated project granted by the European Commission, aimed at developing quality guidelines for molecular in vitro diagnostics and to standardize the preanalytical process in related procedures, was the implementation of External Quality Assessment schemes (EQAs) for the collection, transport and processing of blood samples for RNA and DNA-based analyses [19,20].…”

“…Molecular biology-based procedures have opened new perspectives in diagnosis, prognosis and therapeutic treatments, but the level of standardization of these technologies is often lower than in other areas of laboratory medicine, in particular, despite the increasing number of publication related to the development of standard operating procedures for biobanking [1,2] the effects of pre-analytical factors on analytical performances of molecular test are still poorly investigated [3].…”

Progesterone Receptor Gene (PROGINS) Polymorphism Correlates with Late Onset of Migraine

“…All biological samples were stored at the BioBIM of IRCCS San Raffaele Pisana following the biobanking standard operative procedures (Palmirotta et al, 2011). DNA extraction, PCRs, and sequencing analysis were performed as previously described (Palmirotta et al, 2013b).…”