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A protocol for urine collection and storage prior to DNA methylation analysis.

Author(s): Bosschieter J, Bach S, Bijnsdorp IV, Segerink LI, Rurup WF, van Splunter AP, Bahce I, Novianti PW, Kazemier G, van Moorselaar RJA, Steenbergen RDM, Nieuwenhuijzen JA

Publication: PLoS One, 2018, Vol. 13, Page e0200906

PubMed ID: 30142219 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to investigate the stability of DNA in native urine and urine preserved with EDTA and Urine Conditioning buffer during storage at room temperature, 4˚C, -20˚C, and -80˚C. Urine was collected from three healthy volunteers and immediately divided into four aliquots. One aliquot from each volunteer was used for immediate DNA extraction. EDTA and Urine Conditioning Buffer were added to one aliquot each. The preserved aliquots and the remaining aliquot were then divided for storage at room temperature, 4˚C, -20˚C, and -80˚C. DNA was extracted from 10 mL of urine using the Quick-DNA Urine Kit after 1, 2, 7, and 28 days of storage. DNA was stored at -20˚C until analysis. DNA was bisulfite-converted using the EZ DNA methylation kit and amplification of β-actin (ACTB) was determined by real-time RT PCR. No statistical analysis was performed.

   Summary of Findings:

   Without additives, ACTB levels decreased with storage at room temperature for all time-points and at 4˚C for 7 days or more but were unaffected by storage for up to 28 days at -20˚C or -80˚C. The addition of EDTA or Urine Conditioning Buffer stabilized ACTB levels in urine stored at room temperature and 4˚C. While the urine conditioning provided more stability than EDTA when specimens were stored at room temperature for more than 7 days, there was no difference for shorter durations or when stored at 4˚C.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Frozen
   • Other Preservative
   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	EDTA Frozen None (fresh) Refrigeration Urine conditioning buffer
   Real-time qPCR Specific	Targeted nucleic acid	ACTB
   Storage	Storage duration	1 day 2 days 7 days 28 days
   Storage	Storage temperature	Room temperature 4˚C -20˚C -80˚C

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2. Study Purpose

   This study investigated the stability of ACTB and hypermethylated DNA in unpreserved urine and urine preserved with EDTA and/or PenStrep. Urine was collected from ten patients with bladder cancer and nine patients with non-small cell lung cancer (NSCLC) prior to surgery. Urine specimens were immediately divided into nine aliquots. One aliquot from each volunteer was used for immediate DNA extraction. EDTA (final concentration 40 mM) and PenStrep (final concentration 20 µL/mL) were added alone or in combination to two aliquots per treatment and the remaining two aliquots were left untreated. The aliquots were stored at 4˚C and room temperature (one aliquot each) for 7 days. DNA was extracted from 4-10 mL of urine using the Quick-DNA Urine Kit and stored at -20˚C until analysis. DNA concentrations were determined in some specimens using the Qubit dsDNA kit. DNA was bisulfite-converted using the EZ DNA methylation kit and amplification of β-actin and methylation-specific RASSF1A were determined by real-time RT PCR. For each condition, the change in CT value (ΔCT) with respect to control, log fold change in ACTB, and the correlation were calculated.

   Summary of Findings:

   There was no difference in the ΔCT values for ACTB and RASSF1A between specimens from patients with NCSLC and those with bladder cancer, thus the groups were combined. The log fold change in ACTB with storage at 4˚C for 7 days compared to 0 days was unaffected by the addition of EDTA and/or PenStrep; however, the log fold change in ACTB from day 0 to 7 remained close to zero in specimens containing EDTA when stored at room temperature, regardless of the addition of PenStrep. In unpreserved specimens or those containing only PenStrep, there was a negative log fold change in ACTB from day 0 to 7. As a consequence the log fold change in ACTB was significantly lower in specimens preserved with EDTA than in specimens without EDTA (P<0.001, both). Further, total DNA as determined flourometrically was lower in specimens without EDTA after room temperature storage for 7 days than 0 days, but specimens stored with EDTA did not show similar reductions. The median ΔCT values for methylated RASSF1A were comparable to those for ACTB, regardless of the storage temperature and condition. The preservation of methylated DNA (RASSF1A) was strongly correlated to that of unmethylated DNA, regardless of storage solution and temperature, but more outliers were observed when stored without EDTA.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Other Preservative
   • None (Fresh)

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Bisulfite conversion assay
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	EDTA None (fresh) Refrigeration
   Biospecimen Aliquots and Components	Biospecimen components	EDTA added PenStrep added EDTA and PenStrep added No additive
   Real-time qPCR Specific	Targeted nucleic acid	ACTB RASSF1A
   Storage	Storage duration	0 days 7 days
   Storage	Storage temperature	4˚C Room temperature

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