一种从PAXgene全血RNA管内提取基因组DNA的方法
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摘要
目的:从同一生物样本同步提取RNA和DNA,能提高样本的利用率,而且对于基因组学、转录组学和表观遗传学检测数据之间的比对和匹配分析也十分重要。本研究在不影响RNA样品制备的前提下,建立一种从PAXgene全血RNA管内提取基因组DNA的方法。方法:取一定量PAXgene全血RNA管血液样本,使用QIAamp DNA试剂盒提取血细胞基因组DNA,系统优化提取过程中的离心参数、洗脱量以及初始血液样本量等实验参数,并对提取的基因组DNA质量进行检测。结果:用PAXgene全血RNA管3 mL血液样本能够提取出8.918±1.100μg基因组DNA,紫外分光光度计检测DNA样品的OD 260/280比值为1.89±0.09,琼脂糖凝胶电泳结果显示DNA样品完整无降解。结论:利用本方法提取的DNA样品能够满足下游DNA芯片、DNA甲基化测序等实验要求。该方法有助于从有限的临床血液样本中获取全面的遗传信息,并且提高后续不同实验方法所生成数据之间的可比性和匹配度。
Objective: Simultaneous extraction of RNA and DNA from the same biological sample can improve the utilization of the sample, and it is also very important for the comparison and analysis of the data of genomics, proteomics and epigenetic detection.This study established a method for extracting genomic DNA from PAXgene blood RNA tube without affecting the preparation of RNA samples. Methods: A certain amount of blood samples was taken and gDNA was extracted from blood cells using the QIAamp nucleic acid purification kit. The centrifugal parameters including centrifugal speed and time, elution volume and initial blood sample volume were optimized in the process of extraction and then the quality of the extracted gDNA was detected. Results: We could extract 8.918±1.100 μg(mean ±SD) gDNA from 3 mL tube-sample with an OD 260/280 ratio of 1.89 ±0.09(mean ±SD). The agarose gel electrophoresis analysis showed that the extracted g DNA was of high molecular weight and integrity. Conclusion: The extracted gDNA is generally able to meet the requirements of downstream experiments such as DNA chip, DNA methylation sequencing. This method is helpful to obtain comprehensive genetic information from the limited clinical blood samples, and improve the comparability and the matching degree between the data generated by different experimental methods.
Objective: Simultaneous extraction of RNA and DNA from the same biological sample can improve the utilization of the sample, and it is also very important for the comparison and analysis of the data of genomics, proteomics and epigenetic detection.This study established a method for extracting genomic DNA from PAXgene blood RNA tube without affecting the preparation of RNA samples. Methods: A certain amount of blood samples was taken and gDNA was extracted from blood cells using the QIAamp nucleic acid purification kit. The centrifugal parameters including centrifugal speed and time, elution volume and initial blood sample volume were optimized in the process of extraction and then the quality of the extracted gDNA was detected. Results: We could extract 8.918±1.100 μg(mean ±SD) gDNA from 3 mL tube-sample with an OD 260/280 ratio of 1.89 ±0.09(mean ±SD). The agarose gel electrophoresis analysis showed that the extracted g DNA was of high molecular weight and integrity. Conclusion: The extracted gDNA is generally able to meet the requirements of downstream experiments such as DNA chip, DNA methylation sequencing. This method is helpful to obtain comprehensive genetic information from the limited clinical blood samples, and improve the comparability and the matching degree between the data generated by different experimental methods.
引文
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[14]陈辉,王云云,刘艳.两种自动化全基因血液样本DNA提取效果的比较[J].感染、炎症、修复,2013,14(2):114-115Chen Hui,Wang Yun-yun,Liu Yan.Comparison of DNA extraction effects of two kinds of automated whole gene blood samples[J].Infection;Inflammation;Repair,2013,14(2):114-115
[15]Wu H,de Gannes M K,Luchetti G,et al.Rapid method for the isolation of mammalian sperm DNA[J].Biotechniques,2015,58(6):293-300
[16]Keshavarz Z,Moezzi L,Ranjbaran R,et al.Evaluation of a Modified DNA Extraction Method for Isolation of Cell-Free Fetal DNA from Maternal Serum[J].Avicenna Journal of Medical Biotechnology,2015,7(2):85-88
[17]Kruh?ffer M,Dyrskj?t L,Voss T,et al.Isolation of Microarray-Grade Total RNA,Micro RNA,and DNA from a Single PAXgene Blood RNA Tube[J].Journal of Molecular Diagnostics,2007,9(4):452-458
[18]Soto J,Rodriguez-Antolin C,Vallespín E,et al.The impact of next-generation sequencing on the DNA methylation-based translational cancer research[J].Translational Research,2015,169:1-18
[19]Zhou Y,Xu LY,Derek M Bickhart,et al.Reduced representation bisulphite sequencing of ten bovine somatic tissues reveals DNA methylation patterns and their impacts on gene expression[J].BMC Genomics,2016,17(1):779-790
[20]Brisotto G,Gennaro A D,Damiano V,et al.An improved sequencingbased strategy to estimate locus-specific DNA methylation[J].Bmc Cancer,2015,15(1):1-10
[2]Huancamamani W,Riveracabello D,Maitamaita J.A simple,fast,and inexpensive CTAB-PVP-silica based method for genomic DNA isolation from single,small insect larvae and pupae[J].Genetics&Molecular Research Gmr,2015,14(3):8001-8007
[3]Roume H,Heintz-Buschart A,Muller E E L,et al.Sequential Isolation of Metabolites,RNA,DNA,and Proteins from the Same Unique Sample[J].Methods in Enzymology,2013,531:219-236
[4]Tolosa J M,Schjenken J E,Civiti T D,et al.Column-based method to simultaneously extract DNA,RNA,and proteins from the same sample[J].Biotechniques,2007,43(6):799-804
[5]Yang S,Fahs A,Feldman C,et al.A reliable and effective method of DNA isolation from old human blood paper cards[J].Springerplus,2012,2(1):1-7
[6]Carrol E D,Salway F,Pepper S D,et al.Successful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysis[J].Bmc Immunology,1983,8(1):1-8
[7]Radpour R,Sikora M,Grussenmeyer T,et al.Simultaneous Isolation of DNA,RNA,and Proteins for Genetic,Epigenetic,Transcriptomic,and Proteomic Analysis[J].Journal of Proteome Research,2009,8(11):5264-5274
[8]Chomczynski P.A reagent for the single-step simultaneous isolation of RNA,DNA and proteins from cell and tissue samples[J].Biotechniques,1993,15(3):536-537
[9]Fujii H,Fujita T.Isolation of Specific Genomic Regions and Identification of Their Associated Molecules by Engineered DNA-Binding Molecule-Mediated Chromatin Immunoprecipitation(en Ch IP)Using the CRISPR System and TAL Proteins[J].Int J Mol Sci,2015,16(9):21802-21812
[10]Soriano-Tárraga C,Jiménez-Conde J,Giralt-Steinhauer E,et al.DNA isolation method is a source of global DNA methylation variability measured with LUMA.Experimental analysis and a systematic review[J].Plos One,2013,8(4):1-8
[11]张宁,王凤山.DNA提取方法进展[J].中国海洋药物,2004,23(2):40-46Zhang Ning,Wang Feng-shan.Advances of DNA extraction methods[J].Chinese Journal of Maring Drugs,2004,23(2):40-46
[12]李王霞,刘光箭,陆华新,等.标本因素对全自动磁珠法全血DNA提取的影响[J].中国输血杂志,2014,27(1):29-31Li Wang-xia,Liu Guang-jian,Lu Hua-xin,et al.The quality of automatic extracted DNA associated with blood sample status[J].Chinese Journal of Blood,2014,27(1):29-31
[13]韩芬霞,杨丽芬.全血DNA提取方法的改进及PCR检测[J].江苏农业科学,2012,40(8):56-57Han Fen-xia,Yang Li-fen.Improvement of whole blood DNA extraction method and PCR detection[J].Jiangsu Agricultural Sciences,2012,40(8):56-57
[14]陈辉,王云云,刘艳.两种自动化全基因血液样本DNA提取效果的比较[J].感染、炎症、修复,2013,14(2):114-115Chen Hui,Wang Yun-yun,Liu Yan.Comparison of DNA extraction effects of two kinds of automated whole gene blood samples[J].Infection;Inflammation;Repair,2013,14(2):114-115
[15]Wu H,de Gannes M K,Luchetti G,et al.Rapid method for the isolation of mammalian sperm DNA[J].Biotechniques,2015,58(6):293-300
[16]Keshavarz Z,Moezzi L,Ranjbaran R,et al.Evaluation of a Modified DNA Extraction Method for Isolation of Cell-Free Fetal DNA from Maternal Serum[J].Avicenna Journal of Medical Biotechnology,2015,7(2):85-88
[17]Kruh?ffer M,Dyrskj?t L,Voss T,et al.Isolation of Microarray-Grade Total RNA,Micro RNA,and DNA from a Single PAXgene Blood RNA Tube[J].Journal of Molecular Diagnostics,2007,9(4):452-458
[18]Soto J,Rodriguez-Antolin C,Vallespín E,et al.The impact of next-generation sequencing on the DNA methylation-based translational cancer research[J].Translational Research,2015,169:1-18
[19]Zhou Y,Xu LY,Derek M Bickhart,et al.Reduced representation bisulphite sequencing of ten bovine somatic tissues reveals DNA methylation patterns and their impacts on gene expression[J].BMC Genomics,2016,17(1):779-790
[20]Brisotto G,Gennaro A D,Damiano V,et al.An improved sequencingbased strategy to estimate locus-specific DNA methylation[J].Bmc Cancer,2015,15(1):1-10