Effects of different storage durations on DNA quality of biopsy specimens with novel ultrasound processing

doi:10.12307/2025.018

Abstract

Abstract: BACKGROUND: The technique of ultrasound processing is widely used for molecular biological analysis. It is of great significance to study the DNA quality of tissue with different storage years under new ultrasonic treatment for further specimen quality control of molecular detection.
OBJECTIVE: To explore the effects of different storage durations on DNA quality in specimens with ultrasound processing to investigate the optimal storage time for molecular tests.
METHODS: Forty specimens of breast biopsy were collected and paraffin specimens were prepared by ultrasonography. These specimens were divided into four groups based on their storage periods: < 1 year, 1-3 years, > 3-5 years, and > 5 years, which contained 10 cases in each group. Paraffin specimens were sliced; each slice was 3 μm thick; 10-15 slices were taken, and DNA was extracted. The mass concentration of DNA was examined by Nanophotometer N60 ultra-micro spectrophotometer and Qubit 4.0 fluorometer. The purity of the DNA was analyzed by the ratio of A260/A280. DNA fragment integrity was measured by capillary electrophoresis (Qsep 100) to evaluate the quality of the DNA fragments.
RESULTS AND CONCLUSION: The mean values of A260/A280 in the four groups were between 1.8 and 2.0, meeting the requirements of tests, without significant differences. The mean values of DNA mass concentration (Qubit concentration) were 30.39, 14.33, 2.52, and 1.95 ng/μL, respectively. The mean values of the N/Q were 6.48, 14.18, 24.56, and 29.86. The mean values of DNA were: 5.64, 1.76, 1.24, and 0.80. The percentage of large DNA fragments averaged 56.08%, 17.72%, 12.68%, and 7.90%. Moreover, the Ct values of the internal control detected by PCR were 15.32, 17.09, 18.39, and 21.24. The three other groups exhibited significantly lower DNA concentration, higher N/Q ratios, decreased DNA quality and percentage of large fragments, and increased values of Ct, compared with the group of within 1 year of storage (P < 0.05). The experimental results suggested that for novel ultrasound processed biopsy specimens, we should prioritize samples stored within 1 year for molecular testing. Samples stored within 3 years can also meet the requirements of second-generation sequencing and other tests. Samples stored within 5 years can only be attempted to carry out PCR. Samples stored for more than 5 years were not recommended to carry out molecular tests.

Key words: ultrasonic treatment, storage duration, DNA quality, fragment integrity, degree of degradation, second-generation sequencing

CLC Number:

Shi Chenxi, Zhu Weidong, Li Sanen, Li Xiuming, Shi Feng, Ding Yayun. Effects of different storage durations on DNA quality of biopsy specimens with novel ultrasound processing[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(13): 2655-2660.

Figures/Tables 5

对于DNA质量浓度的质控，目前常见的方法是Nanodrop定量法和Qubit荧光定量法。前者是利用紫外吸收特性获取样本浓度值，如果DNA样本中混有RNA、降解核酸及游离核苷酸等杂质时，定量数据会出现较大偏差。Qubit荧光定量法采用特异荧光染料，可以特异检测目标分子浓度，即使模板有游离核苷酸或降解核苷酸等杂质，检测结果也不受影响。因此，Qubit荧光定量法更加准确，有利于二代测序精准建库[17]。对于核酸质量要求较高的检测平台，如Sanger测序及高通量测序，建议使用敏感度特异性更高的Qubit荧光定量法检测，而Nanodrop可用来评价DNA的纯度。 Nanodrop检测4组DNA质量浓度均值分别为187.80，159.62，58.02，56.45 ng/μL。实验结果表明，存储时间< 1年组与1-3年组样本DNA质量浓度接近，无统计学差异；而存储时间> 3年的两组样本DNA质量浓度明显下降，差异有显著性意义(P < 0.05)，见表1。通过Qubit再次检测了以上样本DNA质量浓度，实验结果显示：4组DNA的质量浓度均值分别为30.39，14.33，2.52，1.95 ng/μL。实验结果表明，与存储时间< 1年组相比，其余3组的DNA质量浓度明显下降，差异有显著性意义(P < 0.05)，见表1。4组DNA总量均值分别为1 519.4，716.7，126.23，97.33 ng。DNA总量显著下降，各组之间差异有显著性意义(P < 0.05)。 4组样本DNA的平均N/Q比值分别为6.29，14.18，24.56，29.86，各组之间差异均有显著性意义(P < 0.05)，见表1。以上结果表明，随着存储年限增长，DNA质量浓度下降，片段降解程度增加。 2.3 快蜡标本存储时长增加，DNA片段完整性降低 2.3.1 快蜡标本存储时长增加，DNA质量数及大片段占比均值明显下降每组随机选取5例标本，提取DNA，通过全自动毛细管电泳进行核酸分析。结果显示，4组样本DNA质量数均值分别为5.64，1.76，1.24，0.80。大片段占比均值分别为56.08%，17.72%，12.68%，7.90%。与< 1年组相比，其余3组DNA质量数及大片段占比均值明显下降，差异有显著性意义(P < 0.05)，见表2。 "

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