Establishment of the First National Standard of NS0 Cell DNA
WU Gang, FU Zhi-hao, CUI Yong-fei, ZHANG Rong-jian, WANG Lan*
National Institutes for Food and Drug Control, Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, Beijing 100050, China
Abstract：OBJECTIVE To prepare the national standard substance for quantitative determination of residual DNA in mouse myeloma(NS0)cells. METHODS NS0 cell DNA was prepared using genomic DNA purification reagents QIAGEN and Genomic-tip 500/G,analyzed for purity and concentration by UV spectrophotometry and agarose gel electrophoresis, and split in 100 microliters and frozen in screw cap tubes. Then five independent laboratories were organized to calibrate the first batch of NS0 DNA national standard using UV spectrophotometry, and evaluated the stability and applicability. RESULTS The prepared national standard substance of NSO DNA was qualified as indicated by A260/A280 between 1.7 and 1.9 and a single specific band in agarose gel electrophoresis. The standard substance was calibrated for 90 times by the five laboratories,and the results showed a geometric mean concentration of 87.5 μg·mL-1, the 95% confidence interval of the geometric mean concentration in a single determination was 86.6-88.5 μg·mL-1. Short-term stability experiments showed that there was no significant change in the standard DNA concentration and A260/A280 ratio after being stored at 4 ℃ for 4 m. The results of storage stability test at -20 and -70 ℃ for 1 year revealed that there was no significant change in the standard DNA concentration and A260/A280 ratio, and the electrophoresis strip was single, so the standard substance was stable at -20 ℃. In applicability studies using the NS0 DNA standard substance, the real-time PCR had high sensitivity up to 0.003 pg of DNA with good linearity (r2＞0.999) in the content range of 3 fg·μL-1-300 pg·μL-1. CONCLUSION The prepared standard substance with batch number of 330002-201701 and DNA concentration of 87.52 μg·mL-1 is qualified in all tests and may be used as national standard substance for residual DNA assay by real-time PCR method.
武刚, 付志浩, 崔永霏, 张荣建, 王兰. NS0细胞DNA首批国家标准品的建立[J]. 中国药学杂志, 2020, 55(5): 389-395.
WU Gang, FU Zhi-hao, CUI Yong-fei, ZHANG Rong-jian, WANG Lan. Establishment of the First National Standard of NS0 Cell DNA. Chinese Pharmaceutical Journal, 2020, 55(5): 389-395.
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