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A quantitative comparison of urine centrifugation and filtration for the isolation and analysis of urinary nucleic acid biomarkers.

Author(s): Djomnang LK, Li C, Mzava O, Cheng AP, Chang A, Lenz JS, Suthanthiran M, Lee JR, Dadhania DM, De Vlaminck I

Publication: Sci Rep, 2024, Vol. 14, Page 10872

PubMed ID: 38740837 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the yield, fragment size profile, distribution by cell/tissue of origin, and microbial DNA abundance in urinary cfDNA isolated from centrifugation supernatants and filtrate. A total of 28 mid-stream clean catch urine specimens were collected from sixteen kidney allograft recipients (11 men and 5 women). Urine was stored for up to 4 h at 4°C before aliquoting. One aliquot of each specimen was centrifuged at 2000 x g for 30 min while the other was filtered through a 1.6 µm ZRC GF filter.  cfDNA was extracted from supernatants and filtrates using the QIAamp Circulating Nucleic Acid Kit and quantified using the Qubit dsDNA HS Assay Kit. For sequencing, cfDNA was bisulfite converted before extraction using the Norgen Urine Cell-Free Circulating DNA Purification Midi Kit, library preparation using the SRSLY Pico-Plus DNA NGS Library Preparation Base Kit, and sequencing using a Nextseq 550 instrument. Sequences were aligned to the human genome using Bismark. Metagenomic abundance was analyzed in a filtered dataset using GRAMMy.

   Summary of Findings:

   Average cfDNA yields were comparable in urinary fluid obtained by centrifugation or filtration (19.95 ng/ml and 18.86 ng/ml, respectively). Further, nuclear and mitochondrial cfDNA fragment length profiles were similar for centrifugation-based supernatants and filtrates. Analysis of next-generation sequencing data found that the proportion of cfDNA that was assigned to each tissue of origin or cell type of origin was similar between supernatants and filtrates. Supernatants and filtrates also had comparable microbial abundance at the genus level, and Bray-Curtis dissimilarity, and Simpson diversity index. The authors conclude that filtration of urine specimens is an acceptable alternative to centrifugation for cfDNA analysis of urinary fluid.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Filtration	Filtered through a 1.6 µm filter Centrifuged at 2000 x g for 30 min
   Biospecimen Aliquots and Components	Centrifugation	Centrifuged Not centrifuged

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2. Study Purpose

   This study compared RNA purity, total RNA yield, and levels of six mRNAs in cells/debris isolated from urine by centrifugation or filtration. A total of 28 mid-stream clean catch urine specimens were collected from sixteen kidney allograft recipients (11 men and 5 women). Urine was stored for up to 4 h at 4°C before aliquoting. One aliquot of each specimen was centrifuged at 2000 x g for 30 min while the other was filtered through a 1.6 µm ZRC GF filter.  RNA was extracted from isolated cells/debris using the RNeasy Mini Kit. Purity and yield were analyzed by NanoDrop. RNA was reverse transcribed using the TaqMan Reverse Transcription Kit, preamplified using custom primers, and quantified by real-time PCR. 

   Summary of Findings:

   RNA purity (A260/A280) and mean CT values for 18S rRNA, TGFβ1, CD3E, IP-10, Pal1, and BKV VP1 were all comparable between urinary cells isolated by centrifugation or filtration. However, the concentration and total yield of RNA were higher when cells were obtained by centrifugation compared to filtration (34.1 ± 32.3 ng/μl versus 23.7 ± 23.8 ng/μl and 1.14 ± 1.42 μg versus 0.70 ± 0.08 μg, respectively). Correlations in CT values for the six mRNAs between cells obtained by centrifugation and filtration ranged from modest to very strong (r=0.50-0.94, P<0.05 all). The T-cell mediated rejection (TCMR) diagnostic signature that was calculated based on the expression of three genes was comparable for cells obtained by centrifugation and filtration. The authors conclude that filtration is an acceptable alternative to centrifugation for analysis of mRNA from urinary cells.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   RNA	Next generation sequencing
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Filtration	Filtered through a 1.6 µm filter Centrifuged at 2000 x g for 30 min
   Biospecimen Aliquots and Components	Centrifugation	Centrifuged Not centrifuged

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