1. Effects of sample preservation conditions on DNA isolation.
- Author
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Akdeniz, Fatma Tuba, Akbulut, Zeynep, Aru, Başak, Yanıkkaya Demirel, Gülderen, Vayvada, Mustafa, Kalamanoğlu Balcı, Merih, Yeğinsu, Ali, Kutlu, Cemal Asım, Terzioğlu, Gökhan, Baktemur, Ebru, Suakar, Öznur, and Kuşkucu, Ayşegül
- Subjects
NUCLEIC acid isolation methods ,DNA ,BLOOD collection ,BLOOD sampling ,RNA analysis - Abstract
Aim: Even though quality of DNA analysis depends on how blood samples are stored, methodological differences also affect quality of analysis. In this study, we isolated DNA from blood samples stored in different conditions (commercial RNA preservation solution, mononuclear cells isolated with density gradient and nucleated cells obtained through whole blood lysis). Method: Blood samples were collected from 11 patients who underwent lung transplantation, taken at 3 different time points: before transplant, 24 hours and 7 days after the procedure. 33 blood samples; stored in commercial RNA preservation solution or isolated mononuclear cells by either density gradient separation or lysing erythrocytes were stored at -80 °C for a year. DNA isolation was performed and samples were evaluated for DNA quality with 260/280 absorbance ratios measured in spectrophotometry. Results: First run of DNA analysis was performed on 18 whole blood samples stored in commercial RNA preservation solution and isolated with 3 different methods: DNA isolation robot, commercial kits, and manual DNA isolation methods (extraction from TRIzol and CTAB-DTAB). DNA analysis was performed on 15 samples' which cells were separated either by density gradient or lysing erythrocytes isolated by 2 different manual methods: extraction from TRIzol and CTAB-DTAB. Among all storage conditions and DNA isolation methods, DNA extraction from TRIzol with isolated cells (either with density gradient or erythrocytes lysis) was found most successful. Conclusion: For molecular analysis, it is important to do validation and verification at every step from the beginning of sample collection. In this study, we got best results with extracting DNA from TRIzol with mononuclear cells stored in Voluven + DMSO among all blood samples. We expect that our methodological approach for validation of pre-analytic phase of this study to be inspiring for research or diagnostic purposes. [ABSTRACT FROM AUTHOR]
- Published
- 2016