Preparation of the first national reference standard for determination of HEK293 cell DNA content
Objective To prepare a national reference standard for the quantification of HEK293 cell DNA content,so as to provide a support for the determination of residual DNA in HEK293 cells in the industry.Methods HEK293 cell DNA prepared using Genomic-tip 500/G and genomic DNA purification reagents was used as source materials,and the purity and content were assessed using ultraviolet spectrophotometry and agarose gel electrophoresis.After dilution to app-roximately 100 ng/μL,the DNA was aliquoted at 160 μL/tube.Five different laboratories were organized for collabora-tive calibration by using ultraviolet spectrophotometry,and the stability and applicability were evaluated.Results The HEK293 cell DNA national reference standard exhibited A260/A280 ratios between 1.8 and 2.0 and displayed a single band on electrophoresis,meeting the specified criteria.Collaborative calibration across five laboratories yielded 78 valid data points with an average content of 104.8 ng/μL,a relative standard deviation(RSD)of 4.2％.The 95％confidence inte-rval for the mean was 103.8-105.8ng/μL,and the 95％reference range for single measurements was 96.0-113.6ng/μL.The average confidence limit rate was 1.0％,and the recommended storage condition was-80 ℃.Applicability studies were conducted using two different models of fluorescence quantitative PCR instruments.The reference standard exhibited good applicability within the range of 0.3-3 000 pg/reaction,with amplification efficiencies of 101％and 95％,andR2 values of 0.999 2 and 0.999 5 for the standard curves,respectively.Conclusion This batch of HEK293 cell DNA national reference standard meets all required specifications and can be utilized as a national reference standard for fluorescence quantitative PCR detection,with a certified content of 104.8 ng/μL,assigned batch number 270039-202301.
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