Recommended Procedure for DNA Extraction from Amniotic Fluid to Genotype Assay of Thalassemia Before and Af-ter Amniotic Fluid Cell Culture
Chen Xiaofang
Ye Wanting
Lin Yuhong
Abstract：Objective:To standardize the procedure of DNA extraction from amniotic fluid(AF),so as to provide referable suggestions and data for the hospitals or institutions that will conduct the genotype of thalassemia by amniotic fluid.Methods:Collecting samples of AF,using TIANamp Micro DNA Kits(Tiangen Biochemical Technology,Beijing,China)to test,to determine the effect on the genotype of thalassemia with AF(PCR reverse dot blot assay)by controlling the storage temperature,time,sample volume and maternal cell contamination(MCC).Results:DNA concentration of 2 mL amniotic fluid was(26.36±2.85)%and the absorbance(OD260/280)was(1.79±0.01).Room temperature,4℃or-20℃for 24 h,and the concentrations all reached the test standards of the Genotype of Thalassemia.The concentration 0,24,48 and 72 h stored at 4℃was 26.32%,26.35%,25.98%and 25.45%,respectively.After cell culture,the concentration of DNA was 205.10%and OD280/260 was 2.06 by taking 20%of 25 mL culture bottle full.The thalassemia genotypes of amniotic fluid cells(DNA template about 50 ng)contaminated with 0.08%or less of whole blood were not affected,while the thalassemia genotypes of amniotic fluid cells with 0.1%or more of whole blood were affected.Conclusion:By controlling the storage time of amniotic fluid cells,sample volume,reducing maternal cell contamination rate and using cultured amniotic fluid cell can improve the efficiency of DNA extraction using Tiangen kit,to further ensure the accuracy of amniotic cell thalassemia genotyping test.
Keywords：ThalassemiaAmniotic fluidDNA extractionPrenatal diagnosis
Publication Date：2025-01-14
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：4( 70-73 )