Abstract��
OBJECTIVE To prepare the national standard substance for quantitative determination of residual DNA in CHO cells. METHODS CHO cell DNA was prepared using QIAGEN Genomic-tip 500/G and genomic DNA purification reagents and analyzed for purity and concentration by UV spectrophotometry and agarose gel electrophoresis. Then the standard substance of CHO DNA was calibrated collaboratively, and evaluated for stability and applicability. RESULTS The prepared national standard substance of CHO 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 six laboratories, and the results showed a geometric mean concentration of 93.63 ��g��mL-1 (95% confidence interval 92.86-94.40 ��g��mL-1). The 95% confidence interval of the geometric mean concentration in a single determination was 86.51-100.89 ��g��mL-1. The mean confidence limit rate was 0.81%. The DNA concentration was stable after storage at -20, 4, 25 and 37 �� for 4 months, but A260/A280 was decreased when stored at 37 �� for 4 months. The electrophoresis results showed a single band after storage at -20, 4 and 25 �� for 4 months, but showed degradation after storage at 37 �� for 4 months. The long term stability test revealed that the DNA concentration and purity were stable after storage at -20 �� for 12 months. In applicability studies using the CHO DNA standard substance, the fluorescence method showed good linearity (r>0.990 0) in the concentration range of 0.781-100 ng��mL-1, with RSD of less than 10%. The real-time PCR had high sensitivity up to 10-2pg of DNA with good linearity (r>0.990 0) in the content range of 10-2-103 pg, and the melting curve showed a single peak. CONCLUSION The prepared standard substance with batch number of 270026-201101 and DNA concentration of 93.63 ��g��mL-1is qualified in overall tests and may be used as national standard substance for residual DNA assay by fluorescence and real-time PCR methods.
WANG Lan,
GAO Kai,
FAN Wen-Hong etc
.Preparation of National Standard Substance of CHO Cell DNA[J] Chinese Pharmaceutical Journal, 2013,V48(1): 68-68
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2013, Vol. 48 