DNA extraction from long-term stored urine.
Author(s): Hilhorst M, Theunissen R, van Rie H, van Paassen P, Tervaert JW
Publication: BMC Nephrol, 2013, Vol. 14, Page 238
PubMed ID: 24168031 PubMed Review Paper? No
Purpose of Paper
This paper assessed the concentration of cell-free DNA (cfDNA) in freeze-dried urine supernatants using two different methods and evaluated if cfDNA concentration was correlated with proteinuria (assessed prior to storage) or the duration of storage at room temperature (6-28 years). cfDNA was isolated from 24-h urine specimens obtained from patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis with pauci-immune necrotizing crescentic glomerulonephritis.
Conclusion of Paper
DNA was obtained from 46 of the 53 24-h urine specimens collected. The mean concentration of DNA in these 46 specimens was 258.7 ng/μL (range 33.2-529), and the mean purity (absorbance 260/280) was 1.81 when quantified by spectrophotometry. In eleven specimens PicoGreen was also used to assess the concentration of double-stranded DNA and the mean was determined to be 23.35 ng/μL. DNA concentration was not correlated with proteinuria (assessed prior to storage) (R2 = 0.02; P = 0.34) or storage duration (R2 = 0.08; P = 0.55). Polymorphisms were detected in urine specimens from males and female patients at a comparable percentage (87.5% versus 54.5%, respectively; P = 0.8).
Studies
-
Study Purpose
This study assessed the concentration of cell-free DNA (cfDNA) in freeze-dried urine supernatants using two different methods and evaluated if cfDNA concentration was correlated with proteinuria (accessed prior to storage) or the duration of storage at room temperature (6-28 years). cfDNA was isolated from urine specimens that were collected over 24 h from fifty-three patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis with pauci-immune necrotizing crescentic glomerulonephritis. Urine was filtered (details not provided) and then dialyzed and concentrated using a Milipore Proflux M12 device. The concentrated urine was frozen in liquid nitrogen and dried in a Beta 1–8 LD freeze dryer before storage at room temperature for 6-28 years (average of 16 years). Urine was rehydrated in MilliQ overnight at 4°C and centrifuged at 10000 g for 10 min before DNA was extracted using the High Pure PCR Template Preparation Kit. DNA concentration and purity were assessed using a NanoDrop spectrophotometer, and double-stranded DNA was quantified in eleven specimens using the Quant-iT Picogreen dsDNA assay. Proteinuria was assessed by an unspecified method prior to specimen storage. The CTLA-4 +49 polymorphism was analyzed by PCR.
Summary of Findings:
DNA was obtained from 46 of the 53 24-h urine specimens collected. The mean concentration of DNA in these 46 specimens was 258.7 ng/μL (range 33.2-529), and the mean purity (absorbance 260/280) was 1.81 when quantified by spectrophotometry. In eleven specimens PicoGreen was also used to assess the concentration of double-stranded DNA and the mean was determined to be 23.35 ng/μL. DNA concentration was not correlated with proteinuria (assessed prior to storage) (R2 = 0.02; P = 0.34) or storage duration (R2 = 0.08; P = 0.55). The CTLA-4 +49 polymorphism was detected in urine specimens from males and female patients at a comparable percentage (87.5% versus 54.5%, respectively; P = 0.8).
Biospecimens
Preservative Types
- Other Preservative
Diagnoses:
- Other diagnoses
Platform:
Analyte Technology Platform DNA Spectrophotometry Protein Clinical chemistry/auto analyzer DNA PCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Patient gender Female
Male
Biospecimen Aliquots and Components Biospecimen components Unspecified range of proteinuria
Storage Time at room temperature 6-28 years (freeze-dried)