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Impact of Collection Volume and DNA Extraction Method on the Detection of Biomarkers and HPV DNA in First-Void Urine.

Author(s): Téblick L, Van Keer S, De Smet A, Van Damme P, Laeremans M, Rios Cortes A, Beyers K, Vankerckhoven V, Matheeussen V, Mandersloot R, Floore A, Meijer CJLM, Steenbergen RDM, Vorsters A

Publication: Molecules, 2021, Vol. 26, Page

PubMed ID: 33915837 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to investigate the potential impact of collection tube volume, DNA extraction method (inclusion of a filtration step, centrifugation step, and kit type) and processing delays on real-time PCR-quantified DNA levels, HPV risk assay results, and amplification of methylation-specific ACTB in urine collected from women with diagnosed with high-risk HPV. Twenty-five women (19-62 years old) with high-risk human papillomavirus (HPV) self-collected a first-void urine specimen into 4, 10 and 20 mL Colli-Pee collection tubes that contained Urine Conservation Medium spiked with phocine herpesvirus 1 (PhHV-1) DNA. The three specimens were collected in random order at an interval of 2:30-4:26. Urine was mailed back to University of Antwerp (0-12 days) where they were aliquoted and stored at -80°C for 1-2 months. Urine was thawed (method unspecified) and DNA was extracted from urine using five different methods: i.) Amicon filtration followed by a modification of the NucliSENS EasyMag Kit (aliquots from 20 mL tubes), ii.) the NucliSENS EasyMag Kit (aliquots from 4 mL, 10 mL and 20 mL tubes), iii.) centrifugation at 800 g for 10 min followed by resuspension of the pellet and extraction with the NucliSENS EasyMag Kit (aliquots from 10 mL and 20 mL tubes), iv.) centrifugation at 3000 g for 10 min followed by resuspension of the pellet and extraction with the NucliSENS EasyMag Kit (aliquots from 10 mL tubes), and v.) centrifugation at 800 g for 10 min followed by resuspension of the pellet and extraction with the QIAamp DNA Blood Mini Kit (aliquots from 20 mL tubes). DNA was quantified by PCR amplification of GAPDH and PhHV-1. HPV genotyping was performed using the real-time PCR-based HPV-Risk assay. DNA was bisulfite converted using the EZ DNA Methylation Kit and ACTB methylation was evaluated by methylation-specific PCR.

   Summary of Findings:

   Cq values were comparable for the internal control among specimens extracted using the same method, regardless of collection tube volume.  However, Cq values for the internal control were significantly affected by extraction method, with significantly lower Cq values observed after DNA extraction using the Amicon filter coupled with the EasyMag Kit compared to all other extraction methods evaluated (P<0.01, all).  Extraction method also affected Cq values of GAPDH, the HV risk assay, and the methylation-specific ACTB assay; although only DNA extracted from urine specimens with the EasyMag Kit generated consistently higher Cq values compared to the other extraction methods evaluatedNonetheless, Cq values were significantly correlated between each of the markers regardless of extraction method. The GAPDH assay and methylated ACTB Cq values were comparable among specimens collected in 4, 10 and 20 mL tubes when extraction was with EasyMag Kit (the only kit used for all collection tube volumes). In urine specimens that underwent DNA extraction with the EasyMag Kit, 8, 11, and 9 of the 25 specimens evaluated were HPV-risk positive in aliquots from 4, 10 and 20 mL collection tubes, respectively, but differences were not significant (P>0.14). GAPDH Cq values were significantly higher among urine aliquots collected in 20 mL tubes than in 10 mL tubes when urine was centrifuged at 800 g prior to extraction with the EasyMag Kit while none of the other markers displayed volume dependent differences in Cq values. Fewer urine specimens were HPV-risk positive when they were collected in 20 mL collection tubes compared to when 10 mL tubes were used when urine was centrifuged at 800 g prior to DNA extraction with the EasyMag Kit (10 versus 13 of 25, significance not evaluated). The duration of time from urine collection to freezing (1-12 days) did not affect Cq values of GAPDH, the HPV risk assay, or methylated ACTB, but Cq values for phHV-1 increased by 0.19 with each passing day between urine collection and frozen storage (P=0.017).

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Frozen

   Diagnoses:

   • Other diagnoses

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Aliquot size/volume	4 mL 10 mL 20 mL
   Biospecimen Aliquots and Components	Centrifugation	Centrifuged Not centrifuged Multiple speeds compared
   Real-time qPCR Specific	Targeted nucleic acid	Methylated ACTB HPV GAPDH
   Analyte Extraction and Purification	Analyte isolation method	Amicon filtration followed by a modification of the NucliSENS EasyMag kit NucliSENS EasyMag Centrifuged at 800 g for 10 min followed by resuspension of the pellet and extraction with NucliSENS EasyMag Centrifuged at 3000 g for 10 min followed by resuspension of the pellet and extraction with NucliSENS EasyMag Centrifuged at 800 g for 10 min followed by resuspension of the pellet and extraction with QIAamp DNA Blood Mini kit
   Storage	Time at room temperature	0-12 days

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