Effects of Centrifugation and Washing of Freeze-Thawed Blood on Isolated Dna Characteristics

Abstract

DNA isolations from the whole blood are commonly performed to obtain DNA for molecular research and diagnostics. Generally, blood samples are taken into anticoagulant tubes and stored in deep freezers until DNA isolation. In fresh blood, pretreatments or leukocytes preparations can be performed and suggested for advanced DNA isolation. However, similar applications in freeze-thawed blood (FTB) have not been shown yet. In the study, centrifugation and washing of FTB were applied as pretreatment before DNA isolations, and their effects on isolated DNA characteristics including DNA integrity, quality, quantity, mitochondrial (mt), and nuclear (n) DNA levels were investigated. Microscopic and flow cytometric analyses were used to check leukocyte integrity in FTB. Spectrophotometric analysis was carried out to determine DNA quality and quantity in the isolated DNA samples. Real-time PCR analyses were used to check mtDNA/nDNA ratio and DNA integrity at the quantitative level. Cell integrity analyses showed that most of the leukocytes were intact in FTB. Therefore, centrifugation enabled intact leukocytes and nuclear pellets in FTB to be harvested and washed and could be applied as pretreatment before DNA isolations. PBS and water washing of FTB led to obtaining high-quality DNA without changing the nDNA/mtDNA ratio and DNA integrity. TE washing of FTB increased DNA quality and enriched nDNA level about 2-fold without changing DNA integrity. Centrifugation and harvesting of a higher volume of FTB increased isolated DNA yield and quality but decreased DNA integrity and nDNA level. To conclude, the pretreatments of FTB had the advantage to obtain DNA with high-quality and high-quantity and can be used before DNA isolation, but they may affect mtDNA/nDNA ratios and DNA integrity levels. The relevant pre-treatment used in the present study can be used and improved for desired DNA isolation from FTB samples.