Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Comprehensive Evaluation and Application of a Novel Method to Isolate Cell-Free DNA Derived From Bile of Biliary Tract Cancer Patients.

Author(s): Shen N, Zhu B, Zhang W, Nian B, Xu X, Yu L, Ruan X, Chen S, Liu Y, Cao X, Shi X, Li Z, Huang X, Wang X, Chen C, Xiong L, Zhang D, Fu X, Zhang Y

Publication: Front Oncol, 2022, Vol. 12, Page 891917

PubMed ID: 35600407 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the recovery and fragment size distribution of cell-free DNA (cfDNA) isolated from bile specimens using an in-house method (3D-BCF) and four commercial kits. Additionally, the authors investigated the suitability of cfDNA isolated using the 3D-BCF method for mutation detection. Bile specimens were collected from four patients with biliary tumors into BEAVER cell-free DNA tubes. Debris was removed by centrifugation at 1,600 g at 4°C for 10 min followed by 16,000 g at 4°C for 15 min. Bile was aliquoted and stored at -80°C until cfDNA extraction. cfDNA was extracted by an in-house method called 3D-BCF which included proteinase K lysis, precipitation in QIAamp Circulating Nucleic Acid Kit buffer, enrichment using a QIA-quick column, washing in Qiagen buffer and elution in a Qiagen buffer. Additionally, cfDNA was extracted from bile from three patients in triplicate using the BIOG cfDNA Easy Kit, QIAamp DNA Mini Kit, MagMAX Cell-Free DNA Isolation Kit and Norgen Urine Cell-Free Circulating DNA Purification Mini Kit.  DNA was quantified by spectrophotometer and Qubit fluorometer, and fragment size analyzed using an Agilent 2100 bioanalyzer. Mutations were detected by indexed capturing-based sequencing using a 733 gene panel. From the NGS data, 10 single nucleotide variants (SNVs)/insertions/deletions were chosen for PCR followed by Sanger sequencing. Three CNVs were selected for verification by FISH in a tumor specimen from a patient (no further details provided).

   Summary of Findings:

   The 3D-BCF method recovered 58.77-69.17% of the spiked-in DNA regardless of input amount (300, 600 or 900 ng). The recovery was highest for short fragments (>60% for ≤1000 bp), but >40% of the long fragments (6000-8000 bp) that were in the spiked-in DNA were also recovered. From each of the three patients, the cfDNA yield was highest using the in-house 3D-BCF method followed by BIOG cfDNA Easy Kit, QIAamp DNA Mini Kit, MagMAX Cell-Free DNA Isolation Kit and Norgen Urine Cell-Free Circulating DNA Purification Mini Kit. The fragment size profile was similar when extraction was with the 3D-BCF method and BIOG cfDNA Easy Kit. The QIAamp DNA Mini Kit isolated a larger percentage of long (>600 bp) fragments but lost most fragments that were <600 bp. The Norgen Kit recovered most short fragments but lost some of the longer fragments.  A total of 47 mutations were found in bile specimens from three patients. Of the 10 mutations selected for PCR amplification and sequencing, nine were confirmed. The authors postulate the last one may have been missed due to low variant allele frequency. All three CNVs analyzed by FISH were verified.

   Biospecimens

   • Bodily Fluid - Bile

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	DNA sequencing
   DNA	FISH
   DNA	Spectrophotometry
   DNA	Next generation sequencing
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	In-house 3D-BCF method BIOG cfDNA Easy Kit QIAamp DNA Mini Kit MagMAX Cell-Free DNA Isolation Kit Norgen Urine Cell-Free Circulating DNA Purification Mini Kit

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