RESUMO
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 (r20.999) 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.