Rong Tang, Cui Han, Ru Yin, Ping Zhu, Ling Zhu, Yinghui Lu, Chunxia Zheng
{"title":"冷冻保存10年以上全细胞微球提取DNA的质量控制。","authors":"Rong Tang, Cui Han, Ru Yin, Ping Zhu, Ling Zhu, Yinghui Lu, Chunxia Zheng","doi":"10.1089/bio.2021.0052","DOIUrl":null,"url":null,"abstract":"<p><p><b><i>Background:</i></b> Cryopreserved whole blood, all-cell pellets (ACPs), and buffy coats in biobanks are widely used to obtain DNA for genetic testing. However, there are few studies concerning the quality control of DNA extracted from them. Our research aimed to perform quality control of DNA extracted from ACPs after cryopreservation for >10 years. <b><i>Materials and Methods:</i></b> A total of 1377 ACP samples (separated from 3 mL of whole blood) were retrieved from our biobank, where they had been cryopreserved for 10-15 years. Chemagic STAR was used to extract the DNA. Absorbance at A260, A280, and A230 were measured by spectrophotometry, and integrity was analyzed by agarose gel electrophoresis. The quality thresholds for an Illumina Asian Screening Array (ASA) were yields greater than 0.5 μg, concentration of 25-150 ng/μL, A260/280 ratio of 1.6-2.1, and no degradation fragments in the electrophoresis gel. <b><i>Results:</i></b> The median yield of genomic DNA was 54.30 μg (interquartile range [IQR] 35.55-74.64). The median A260/280 and A260/230 ratios were 1.90 (IQR 1.87-1.94) and 1.98 (IQR 1.64-2.41), respectively. In total, 1377 samples (100%) had qualified yields, and 1366 samples (99.20%) had qualified integrity results. Finally, 1328 (96.44%) samples were used for ASA. Of the remaining samples, 34 needed to be repurified, 4 were obtained at an insufficient concentration, and 11 were unqualified for integrity. In addition, we analyzed the influence of hemolysis (90 samples) and clots (102 samples) on the quality of DNA samples. Hemolysis and clotting did not influence yield or integrity, but a significant difference was found in A260/230 compared to normal samples (<i>p</i> < 0.05). Furthermore, the samples (14 samples) with both hemolysis and clots had higher A260/280 (<i>p</i> < 0.05). <b><i>Conclusion:</i></b> ACP samples stored for >10 years at -80°C produced DNA with high quality for use in genetic analysis. Hemolysis and clots in the ACPs led to lower purity, but did not significantly affect yield or integrity.</p>","PeriodicalId":49231,"journal":{"name":"Biopreservation and Biobanking","volume":" ","pages":"211-216"},"PeriodicalIF":1.2000,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Quality Control of DNA Extracted from All-Cell Pellets After Cryopreservation for More Than 10 Years.\",\"authors\":\"Rong Tang, Cui Han, Ru Yin, Ping Zhu, Ling Zhu, Yinghui Lu, Chunxia Zheng\",\"doi\":\"10.1089/bio.2021.0052\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p><b><i>Background:</i></b> Cryopreserved whole blood, all-cell pellets (ACPs), and buffy coats in biobanks are widely used to obtain DNA for genetic testing. However, there are few studies concerning the quality control of DNA extracted from them. Our research aimed to perform quality control of DNA extracted from ACPs after cryopreservation for >10 years. <b><i>Materials and Methods:</i></b> A total of 1377 ACP samples (separated from 3 mL of whole blood) were retrieved from our biobank, where they had been cryopreserved for 10-15 years. Chemagic STAR was used to extract the DNA. Absorbance at A260, A280, and A230 were measured by spectrophotometry, and integrity was analyzed by agarose gel electrophoresis. The quality thresholds for an Illumina Asian Screening Array (ASA) were yields greater than 0.5 μg, concentration of 25-150 ng/μL, A260/280 ratio of 1.6-2.1, and no degradation fragments in the electrophoresis gel. <b><i>Results:</i></b> The median yield of genomic DNA was 54.30 μg (interquartile range [IQR] 35.55-74.64). The median A260/280 and A260/230 ratios were 1.90 (IQR 1.87-1.94) and 1.98 (IQR 1.64-2.41), respectively. In total, 1377 samples (100%) had qualified yields, and 1366 samples (99.20%) had qualified integrity results. Finally, 1328 (96.44%) samples were used for ASA. Of the remaining samples, 34 needed to be repurified, 4 were obtained at an insufficient concentration, and 11 were unqualified for integrity. In addition, we analyzed the influence of hemolysis (90 samples) and clots (102 samples) on the quality of DNA samples. Hemolysis and clotting did not influence yield or integrity, but a significant difference was found in A260/230 compared to normal samples (<i>p</i> < 0.05). Furthermore, the samples (14 samples) with both hemolysis and clots had higher A260/280 (<i>p</i> < 0.05). <b><i>Conclusion:</i></b> ACP samples stored for >10 years at -80°C produced DNA with high quality for use in genetic analysis. Hemolysis and clots in the ACPs led to lower purity, but did not significantly affect yield or integrity.</p>\",\"PeriodicalId\":49231,\"journal\":{\"name\":\"Biopreservation and Biobanking\",\"volume\":\" \",\"pages\":\"211-216\"},\"PeriodicalIF\":1.2000,\"publicationDate\":\"2022-06-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biopreservation and Biobanking\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1089/bio.2021.0052\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2021/8/25 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q4\",\"JCRName\":\"CELL BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biopreservation and Biobanking","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1089/bio.2021.0052","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2021/8/25 0:00:00","PubModel":"Epub","JCR":"Q4","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
Quality Control of DNA Extracted from All-Cell Pellets After Cryopreservation for More Than 10 Years.
Background: Cryopreserved whole blood, all-cell pellets (ACPs), and buffy coats in biobanks are widely used to obtain DNA for genetic testing. However, there are few studies concerning the quality control of DNA extracted from them. Our research aimed to perform quality control of DNA extracted from ACPs after cryopreservation for >10 years. Materials and Methods: A total of 1377 ACP samples (separated from 3 mL of whole blood) were retrieved from our biobank, where they had been cryopreserved for 10-15 years. Chemagic STAR was used to extract the DNA. Absorbance at A260, A280, and A230 were measured by spectrophotometry, and integrity was analyzed by agarose gel electrophoresis. The quality thresholds for an Illumina Asian Screening Array (ASA) were yields greater than 0.5 μg, concentration of 25-150 ng/μL, A260/280 ratio of 1.6-2.1, and no degradation fragments in the electrophoresis gel. Results: The median yield of genomic DNA was 54.30 μg (interquartile range [IQR] 35.55-74.64). The median A260/280 and A260/230 ratios were 1.90 (IQR 1.87-1.94) and 1.98 (IQR 1.64-2.41), respectively. In total, 1377 samples (100%) had qualified yields, and 1366 samples (99.20%) had qualified integrity results. Finally, 1328 (96.44%) samples were used for ASA. Of the remaining samples, 34 needed to be repurified, 4 were obtained at an insufficient concentration, and 11 were unqualified for integrity. In addition, we analyzed the influence of hemolysis (90 samples) and clots (102 samples) on the quality of DNA samples. Hemolysis and clotting did not influence yield or integrity, but a significant difference was found in A260/230 compared to normal samples (p < 0.05). Furthermore, the samples (14 samples) with both hemolysis and clots had higher A260/280 (p < 0.05). Conclusion: ACP samples stored for >10 years at -80°C produced DNA with high quality for use in genetic analysis. Hemolysis and clots in the ACPs led to lower purity, but did not significantly affect yield or integrity.
期刊介绍：
Biopreservation and Biobanking is the first journal to provide a unifying forum for the peer-reviewed communication of recent advances in the emerging and evolving field of biospecimen procurement, processing, preservation and banking, distribution, and use. The Journal publishes a range of original articles focusing on current challenges and problems in biopreservation, and advances in methods to address these issues related to the processing of macromolecules, cells, and tissues for research.
In a new section dedicated to Emerging Markets and Technologies, the Journal highlights the emergence of new markets and technologies that are either adopting or disrupting the biobank framework as they imprint on society. The solutions presented here are anticipated to help drive innovation within the biobank community.
Biopreservation and Biobanking also explores the ethical, legal, and societal considerations surrounding biobanking and biorepository operation. Ideas and practical solutions relevant to improved quality, efficiency, and sustainability of repositories, and relating to their management, operation and oversight are discussed as well.
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