Effects Of Preservation Solution On The Urine Genomic DNA Quality And Its Application Research

With the rise of the concept of precision medicine,liquid biopsy technology based on peripheral blood cell free DNA(cfDNA)has entered the field of clinical medical detection.Urine,as a source of non-invasive samples,outperforms tissue and blood,especially bladder cancer patients who need repeatedly to monitor cancer progression and therapeutic effect.Unlike serum or plasma collection,urine collection does not require professional facilities or trained professionals.With the development of gene sequencing technology,the detection of molecular markers related to bladder cancer at the molecular level can improve the sensitivity and specificity of early diagnosis of bladder cancer.However,genetic testing has a high requirement for the integrity of DNA samples extracted from urine.This study will focus on the composition of the urine preservation solution,so as to obtain genomic DNA of urine with a higher degree of integrity,which can be applied to the diagnosis of bladder cancer and other diseases and the monitoring of disease progress.In this study,four basic preservation solutions were initially screened using the simulated sample of T24 cell line of bladder cancer.The integrity of genomic DNA extracted after the urine samples with four kinds of preservation solutions were stored for different times was analyzed,and the optimal basic formula 3(2.47 mM EDTA,0.2 mM Tris-HCl,0.5 M LiCl,0.55 M urea,0.17 mM SDS,90 mL anhydrous ethanol,pH 7.0)was selected.On the basis of formula 3,the key components of urea and SDS were gradient tested,a total of 36 formulations were screened.Through the integrity analysis of genomic DNA extracted after the preservation of 0,1,2,4 weeks,the formula ID21(2.47 mM EDTA,0.2 mM Tris-HCl,0.5 M LiCl,0.5 M urea,0.17 mM SDS,90 mL anhydrous ethanol,pH 7.0)was determined to be the optimal formula for urine preservation,and then 71 clinical samples were used for verification.The preservation effect of urine was evaluated by comparing the quality of genomic DNA extracted from clinical samples with and without the addition of ID21 preservation solution and commercial preservation solution.The results showed that the preservation solution proposed in this study greatly improved the total amount and integrity of DNA.After one month of preservation,the amount of DNA extracted from the urine preservation solution added with ID21 was about 6 times of that without the addition of urine preservation solution and 3 times of that with the addition of commercial preservation solution.The ALU-C4 copy number is kept above 10~6 level,which is an order of magnitude higher than the non-added urine preservation solution and the added commercial preservation solution,and can be better applied to clinical and scientific research needs.The 18samples urine preservation liquid formula ID21was applied in the screening project of methylation markers of bladder cancer.After the addition of clinical samples and the storage of stored urine for 0-4 weeks,bladder cancer markers could be effectively detected.Using QMSP technology,the highest combined predictors were determined to be C3:PCDH17+POU4F2+EOMES.