血液样本中DNA与RNA提取效率的研究

doi:10.3969/j.issn.1674-8115.2025.04.010

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (4): 476-486.doi: 10.3969/j.issn.1674-8115.2025.04.010

• 论著 · 技术与方法 • 上一篇下一篇

血液样本中DNA与RNA提取效率的研究

宿星蕾¹^,²^,³, 路平⁴, 彭俊杰⁴, 汪滋民⁵, 宋萍³(), 韩达¹^,²()

^1.上海大学生命科学学院，上海 200444
^2.上海交通大学医学院分子医学研究院，上海 200127
^3.上海交通大学生物医学工程学院，上海交通大学张江高等研究院，上海 200240
^4.复旦大学附属肿瘤医院大肠外科，上海 200032
^5.上海交通大学医学院附属第九人民医院骨科，上海 200011

收稿日期:2024-08-06 接受日期:2024-12-09 出版日期:2025-04-28 发布日期:2025-04-28
通讯作者: 宋萍,韩达 E-mail:songpingsjtu@sjtu.edu.cn;dahan@sjtu.edu.cn
作者简介:宿星蕾（2000—），女，硕士生；电子信箱：suxinglei@shu.edu.cn。
基金资助:
国家重点研发计划(2021YFF1200300);国家自然科学基金(22174094)

Comparison of DNA and RNA extraction efficiency from blood

SU Xinglei¹^,²^,³, LU Ping⁴, PENG Junjie⁴, WANG Zimin⁵, SONG Ping³(), HAN Da¹^,²()

^1.School of Life Sciences, Shanghai University, Shanghai 200444, China
^2.Institute of Molecular Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
^3.School of Biomedical Engineering, Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai 200240, China
^4.Department of Colorectal Surgery, Fudan University Shanghai Cancer Center, Shanghai 200032, China
^5.Department of Orthopedics, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China

Received:2024-08-06 Accepted:2024-12-09 Online:2025-04-28 Published:2025-04-28
Contact: SONG Ping, HAN Da E-mail:songpingsjtu@sjtu.edu.cn;dahan@sjtu.edu.cn
Supported by:
National Key Research and Development Program of China(2021YFF1200300);National Natural Science Foundation of China(22174094)

摘要/Abstract

摘要：

目的·全面评估不同试剂盒及不同方法对血液样本中DNA与RNA提取的效率。方法·收集来自阿尔茨海默病（20例）、纤维化（5例）、结直肠癌患者（108例）及健康个体（12例）的血液样本，共145例。采用柱法试剂盒（Kit A）和核酸提取仪来提取血液中白细胞的基因组DNA（genomic DNA，gDNA）。使用5种试剂盒（Kit B~F），通过柱法（Kit B、E）或磁珠法（Kit C、D、F）对血浆中的游离DNA（cell-free DNA，cfDNA）和游离RNA（cell-free RNA，cfRNA）进行提取。对Kit B提取过程进行优化，包括增加血浆上样体积和延长洗脱孵育时间，并且采用该方案对100例结直肠癌患者血浆样本cfDNA进行提取。使用实时荧光定量PCR（quantitative real-time PCR，qPCR）对提取出的DNA和RNA进行定量分析，比较提取的DNA或RNA分子的数量以评估提取效率，并结合成本及操作时间等进行综合评价。结果·在gDNA提取中，尽管核酸提取仪缩短了操作时间，但Kit A（柱法）提取的DNA分子数的中位数是其25.36倍（P<0.001）。对于cfDNA提取，3种试剂盒（Kit B~D）的总体效率相近，但Kit B（柱法）在低浓度样本中的表现较为突出，其DNA提取量的平均值分别是基于磁珠法的Kit D和Kit C的4.24倍和1.18倍。提取步骤优化后的Kit B进一步提高了cfDNA的提取效率。对比3例样本在不同血浆上样体积下的cfDNA提取量，结果显示大上样体积提取的cfDNA量分别为小上样体积的3.98倍、2.38倍和3.82倍；对100例结直肠癌患者血浆样本cfDNA提取结果表明，该提取方案可在临床样本中稳定提取足够量的cfDNA。在cfRNA提取方面，Kit E（柱法）因其高效、便捷且经济性好，被广泛推荐；其提取的cfRNA含量中位数是Kit F（磁珠法）的5.01倍。比较血浆中cfDNA与cfRNA的拷贝数，结果显示每毫升血浆中cfRNA的拷贝数平均数是cfDNA的27.65倍。结论·Kit A、Kit B和Kit E分别在白细胞gDNA、血浆cfDNA以及血浆cfRNA提取方面表现出更高的效率。然而，尽管Kit E在提取效率和成本上具有优势，其安全性仍需进一步评估。

关键词: 核酸提取, 血浆游离DNA, 血浆游离RNA, 提取试剂盒, 实时荧光定量PCR

Abstract:

Objective ·To comprehensively evaluate the efficiency of different kits and methods for DNA and RNA extraction from blood samples. Methods ·A total of 145 blood samples were collected, including those from patients with Alzheimer's disease (20 cases), fibrosis (5 cases), colorectal cancer (108 cases), and healthy individuals (12 cases). A column-based kit (Kit A) and a nucleic acid extraction instrument were used to extract genomic DNA (gDNA) from leukocytes in the blood. Cell-free DNA (cfDNA) and cell-free RNA (cfRNA) in plasma were extracted using five different kits (Kit B‒F), which employed either column-based (Kit B, E) or magnetic bead-based methods (Kit C, D, F). The extraction process of Kit B was optimized by increasing the plasma sample volume and extending the elution incubation time. Furthermore, this protocol was applied to extracting cfDNA from plasma samples of 100 colorectal cancer patients. Quantitative real-time PCR (qPCR) was used to quantify the extracted DNA and RNA, and the molecular yields were compared to evaluate the extraction efficiency. A comprehensive assessment was conducted, considering factors such as cost and operation time. Results ·In gDNA extraction, although the the operation time was shortened by using the nucleic acid extraction instrument, the median number of DNA molecules extracted using Kit A (column-based method) was 25.36-fold higher than that obtained with the instrument (P<0.05). For cfDNA extraction, while the overall efficiency of the three kits (Kit B‒D) was similar, Kit B (column-based method) showed superior performance in low-concentration samples, with average DNA yields 4.24-fold and 1.18-fold higher than those of Kit D and Kit C (both magnetic bead-based). Optimization of Kit B's extraction protocol further improved cfDNA yield. When comparing three samples, the cfDNA yields from larger plasma input volumes was 3.98-fold, 2.38-fold, and 3.82-fold higher than those from smaller input volumes, respectively. The results of cfDNA extraction from 100 colorectal cancer patients indicated that this extraction protocol reliably extracted sufficient amounts of cfDNA from clinical samples. For cfRNA extraction, Kit E (column-based method) was widely recommended due to its high efficiency, convenience, and cost-effectiveness. The median RNA content extracted using Kit E was 5.01-fold higher than that of Kit F (magnetic bead-based method). Lastly, a comparison of the copy numbers of cfDNA and cfRNA in plasma revealed that the average copy number of cfRNA per milliliter of plasma was 27.65-fold higher than that of cfDNA. Conclusion ·Kit A, Kit B, and Kit E show outstanding performance in leukocyte gDNA extraction, plasma cfDNA extraction, and plasma cfRNA extraction, respectively. However, although Kit E has advantages in extraction efficiency and cost, its safety requires further evaluation.

Key words: nucleicacid extraction, plasma cell-free DNA (cfDNA), plasma cell-free RNA (cfRNA), extraction kit, quantitative real-time PCR (qPCR)

中图分类号:

R446.1

宿星蕾, 路平, 彭俊杰, 汪滋民, 宋萍, 韩达. 血液样本中DNA与RNA提取效率的研究[J]. 上海交通大学学报（医学版）, 2025, 45(4): 476-486.

SU Xinglei, LU Ping, PENG Junjie, WANG Zimin, SONG Ping, HAN Da. Comparison of DNA and RNA extraction efficiency from blood[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(4): 476-486.

图/表 11

表1 qPCR引物序列

Tab 1 Primer sequences for qPCR

Name	Forward (5'→3')	Reverse (5'→3')
Amplicon 1	GCTCTTCCTCTCACATCTTTATTTAACC	TCCACACTGCAGTGTGAACAG
Amplicon 2	GCTCTTCCTCTCACATCTTTATTTAACC	AGAAGTAATCAGATTCTGGAAGAGCTG
Amplicon 3	GAAGGTGAAGGTCGGACAAC	CGAGTTAAAAGCAGCCTGGT

图1 基于qPCR技术制作的2组标准曲线Note： A. Standard curve plotted based on DNA concentration and CT value after qPCR reaction using amplicon 1 with an amplified DNA fragment length of 103 bp. B. Standard curve plotted based on DNA concentration and CT value after qPCR reaction using amplicon 2 with an amplified DNA fragment length of 71 bp. The correlation coefficients (R²) for the standard curves are close to 1 (R²≥0.999), indicating a strong linear relationship between logarithmic value of sample concentration (ng/μL) and CT signal intensity.

Fig 1 Two sets of standard curves based on qPCR technology

图2 白细胞gDNA不同提取方法效率对比Note： A. Schematic of leukocyte gDNA extraction methods, including extraction using Kit A and automated extraction with a nucleic acid extractor. B. Scatter plots of DNA content extracted from samples of 20 patients with Alzheimer's disease using a nucleic acid extractor and a nucleic acid extraction kit respectively. C. Comparison of gDNA extraction efficiency from samples of 20 patients with Alzheimer's disease using a nucleic acid extractor and a nucleic acid extraction kit.

Fig 2 Comparison of the efficiency of various methods for extracting leukocyte gDNA

表2 白细胞gDNA提取试剂盒及核酸提取仪的基本特征

Tab 2 Basic characteristics of leukocyte gDNA extraction kit and nucleic acid extraction instrument

Elution

volume/μL

Indicative price for each reaction/yuan

EX3600 plus automated nucleic acid extractor

Magnetic bead

Whole blood, serum, and plasma

300

50‒100

图3 3种cfDNA提取方法比较Note： A. Schematic of two plasma DNA extraction methods: the magnetic bead-based methods (Kit C and Kit D) and the column-based method (Kit B). B. Comparison of DNA content extracted from 1 mL of plasma using Kit B and Kit D respectively. Plasma samples were obtained from 6 patients with colorectal cancer. C. Comparison of DNA content extracted from 1 mL of plasma using Kit B and Kit C respectively. Plasma samples were obtained from 5 fibrosis patients.

Fig 3 Comparison of three cfDNA extraction methods

表3 血浆cfDNA提取试剂盒基本特征

Tab 3 Basic characteristics of plasma cfDNA extraction kits

Name	Extraction method	Time-consumin/min	Sample type	Applicable volume/μL	Elution volume/μL	Indicative price for each reaction/yuan
Kit B	Adsorption column	60	Plasma, serum, and cell-free body fluid	100‒200	50‒100	60
Kit C	Magnetic bead	70	Serum, plasma, and cell-free body fluid	200‒2 000	30‒50	30
Kit D	Magnetic bead	50	Plasma, serum, urine, pleural fluid, cerebrospinal fluid, and saliva	100‒5 000	50‒100	30

图4 血浆cfDNA提取流程优化及100例结直肠癌样本血浆cfDNA提取Note： A. Schematic of the optimized cfDNA extraction workflow from plasma by increasing the sample loading volume and extending the elution incubation time. B. Analysis of the results of cfDNA extraction using Kit B and optimized extraction process for sample 31 (initial input volumes: 0.3, 0.9, and 1.8 mL) from fibrosis patient and samples 32 and 33 (initial input volumes: 1.5 and 2.1 mL) from colorectal cancer patients. C. Plasma cfDNA content extracted from 100 colorectal cancer patient samples using Kit B.

Fig 4 Optimization of plasma cfDNA extraction process and plasma cfDNA extraction from 100 colorectal cancer samples

图5 血浆中cfDNA受存放时间影响及扩增子长度对DNA分子捕获差异的比较Note：A. cfDNA content in the first elution of colorectal cancer patient samples 32 and 33 at three time points (30 μL elution). B. cfDNA content in both the first and second elutions of colorectal cancer patient samples 32 and 33 at three time points (20 μL elution for the second). C. Schematic of amplicon design: amplicon 1 (103 bp) and amplicon 2 (71 bp). D. qPCR quantification of cfDNA from fibrosis patient sample 31 using the two amplicons (30 μL for the first elution, 20 μL for the second and third).

Fig 5 Comparison of the impact of storage time on plasma cfDNA and the difference in DNA molecule capture based on primer length

图6 血浆中cfRNA提取效率对比Note： A. Schematic comparison of cfRNA extraction methods using column-based and magnetic bead-based techniques. Samples 34-1 and 35-1 represent the first 30 μL elutions, while samples 34-2 and 35-2 represent the second 20 μL elutions. B. Comparison of cfDNA copy number (per microliter of plasma) extracted from the blood plasma of two normal individuals using Kit E and Kit F. The line chart shows that the column-based method is more efficient in extraction. C. Comparison of the log copy numbers of cfRNA extracted using Kit E and Kit F from 1 mL of blood plasma from two normal individuals, with two elutions of DNA content.

Fig 6 Comparison of cfRNA extraction efficiency from plasma

表4 血浆cfRNA提取试剂盒基本特征

Tab 4 Basic characteristics of plasma cfRNA extraction kits

Indicative price for each time/

yuan

Kit E

Adsorption column

Blood, plasma, serum, and lymph

图7 10例样本血浆中cfDNA与cfRNA含量对比

Fig 7 Comparison of cfDNA and cfRNA content in plasma from 10 samples

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血液样本中DNA与RNA提取效率的研究

Comparison of DNA and RNA extraction efficiency from blood

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