Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Establishment of liquid biopsy procedure for the analysis of circulating cell free DNA, exosomes, RNA and proteins in colorectal cancer and adenoma patients.

Author(s): Čeri A, Somborac-Bačura A, Fabijanec M, Hulina-Tomašković A, Matusina M, Detel D, Verbanac D, Barišić K

Publication: Sci Rep, 2024, Vol. 14, Page 26925

PubMed ID: 39506031 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the yield and fragment size of cell-free DNA isolated from plasma of colorectal carcinoma patients using three different kits (QIAamp Circulating Nucleic Acid Kit, QIAamp ccfDNA/RNA Kit and NucleoSpin cfDNA XS Kit). Blood was collected from 11 patients with colorectal carcinoma into CellSave tubes and stored at 4°C for < 6h before plasma separation. Plasma was separated by centrifugation at 1,900 × g for 10 min at 4°C followed by centrifugation of the supernatant at 16,000 × g for 10 min at 4°C. Plasma aliquots were stored for <1 month at -20°C or <6 months at -80°C. cfDNA was isolated from plasma using the QIAamp Circulating Nucleic Acid Kit, QIAamp ccfDNA/RNA Kit and the NucleoSpin cfDNA XS Kit and stored at -20°C until quantification with the Qubit dsDNA HS Assay Kit and fragment size analysis using the High Sensitivity DNA Kit on a Bioanalyzer 2100 instrument.

   Summary of Findings:

   The yield of cfDNA per mL of plasma was significantly higher when extraction was with the QIAamp Circulating Nucleic Acid Kit rather than the QIAamp ccfDNA/RNA Kit or NucleoSpin cfDNA XS Kit (P<0.001). Electrophoretic peaks corresponding to mononucleosomal cfDNA (146-190 bp) were observed in DNA from all 11 specimens when isolation was with the QIAamp Circulating Nucleic Acid Kit and 9 of 11 specimens when isolation was with the QIAamp ccfDNA/RNA Kit but only 2 of 11 specimens when extraction was with the NucleoSpin cfDNA XS Kit. Dinucleosomal (~320 bp) peaks were observed in DNA isolated from 4 specimens when extraction was with the QIAamp Circulating Nucleic Acid Kit and 2 specimens when extraction was with the QIAamp ccfDNA/RNA Kit.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	QIAamp Circulating Nucleic Acid Kit QIAamp ccfDNA/RNA Kit NucleoSpin cfDNA XS Kit

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2. Study Purpose

   This paper compared the yield, size, protein content, levels of exosomal and cytoplasmic proteins and levels of miR-19a-3p and miR-92a-3p in exosomes isolated from plasma using the miRCURY Exosome Serum/Plasma Kit and the Total Exosome Isolation Kit. Blood was collected from 11 patients with colorectal carcinoma into CellSave tubes and stored at 4°C for < 6h before plasma separation. Plasma was separated by centrifugation at 1,900 × g for 10 min at 4°C followed by centrifugation of the supernatant at 3,000 × g for 15 min at 4°C. Plasma aliquots were stored for <1 month at -20°C or <6 months at -80°C. Plasma was thawed on ice and debris was removed by centrifugation at 3,000 × g for 15 min at 4°C. Exosomes were then isolated using a modified protocol of the Total Exosome Isolation Kit (a centrifugation step at 10,000 × g for 20 min at room temperature was performed before the first step) and the miRCURY Exosome Serum/Plasma Kit (first centrifugation was for 10 min). The size and yield of particles were assessed by light scattering using a Zetasizer Nano ZS instrument. Protein content was quantified by bicinchoninic acid (BCA) assay. CD9, CD63, ALIX and TSG101 in exosomes were analyzed by Western blot. RNA was extracted from exosomes isolated with the miRCURY Exosome Serum/Plasma Kit using miRNeasy Serum/Plasma Advanced Kit and from exosomes isolated with the Total Exosome Isolation Kit using the Total Exosome RNA and Protein Isolation Kit. miR-19a-3p and miR-92a-3p were quantified by real-time PCR and normalized to UniSp6.

   Summary of Findings:

   While exosome isolation using the miRCURY Exosome Serum/Plasma Kit resulted in higher mean exosome yields (379.1 x 10¹³ versus 4.7 x 10¹³ particles/mL, P=0.016) and mean protein content (21.1 versus 0.6 ng/mL, P=0.016) than isolation with the Total Exosome Isolation Kit, the mean particle size was also smaller (29.9 versus 86.4 nm, P=0.016). The exosomal markers CD9 and CD63 and the cytoplasmic markers ALIX and TSG101 were observed in all exosomes, but the abundance of the exosomal markers was much higher when isolation was with the miRCURY Exosome Serum/Plasma Kit than the Total Exosome Isolation Kit. The levels of miR-19a-3p and miR-92a-3p were comparable when RNA was extracted from miRCURY-isolated exosomes using the miRNeasy Serum/Plasma Advanced Kit and Total Exosome-isolated exosomes using the Total Exosome RNA and Protein Isolation Kit.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Light scattering
   Morphology	Light scattering
   RNA	Real-time qRT-PCR
   Protein	Colorimetric assay
   Protein	Western blot

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Total Exosome Isolation Kit (RNA with Total Exosome RNA and Protein Isolation Kit) miRCURY Exosome Serum/Plasma Kit (RNA with miRNeasy Serum/Plasma Advanced Kit)
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-19a-3p miR-92a-3p
   Western blot Specific	Targeted peptide/protein	CD9 CD63 ALIX TSG101

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