Establishment of liquid biopsy procedure for the analysis of circulating cell free DNA, exosomes, RNA and proteins in colorectal cancer and adenoma patients
Abstract Liquid biopsy has an underexplored diagnostic potential in colorectal cancer (CRC). Sufficient quantity and quality of its elements (circulating cell-free DNA (ccfDNA), exosomes and exosomal RNA) are essential for accurate results. The present study aims to establish the optimal protocol fo...
Saved in:
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2024-11-01
|
| Series: | Scientific Reports |
| Subjects: | |
| Online Access: | https://doi.org/10.1038/s41598-024-78497-x |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850129150977769472 |
|---|---|
| author | Andrea Čeri Anita Somborac-Bačura Marija Fabijanec Andrea Hulina-Tomašković Marko Matusina Dijana Detel Donatella Verbanac Karmela Barišić |
| author_facet | Andrea Čeri Anita Somborac-Bačura Marija Fabijanec Andrea Hulina-Tomašković Marko Matusina Dijana Detel Donatella Verbanac Karmela Barišić |
| author_sort | Andrea Čeri |
| collection | DOAJ |
| description | Abstract Liquid biopsy has an underexplored diagnostic potential in colorectal cancer (CRC). Sufficient quantity and quality of its elements (circulating cell-free DNA (ccfDNA), exosomes and exosomal RNA) are essential for accurate results. The present study aims to establish the optimal protocol for handling liquid biopsy samples. Samples were obtained by collecting peripheral blood from colorectal adenoma patients in CellSave tubes. Plasma was separated within six hours using differential centrifugation and aliquots stored at – 20/– 80 °C until further processing. Three methods for isolation of ccfDNA, and two combinations of kits for isolation of exosomes and exosomal RNA were tested. The quality and quantity of ccfDNA isolates were evaluated. Exosomes were characterised by determining size, concentration, and total and specific protein content. Expression of chosen microRNAs, miR-19a-3p and miR-92-3p, which have been implicated in CRC progression, were determined. The vacuum-column-based kit showed the highest quantities of isolated ccfDNA (P-value < 0.001). Kits for exosome isolation significantly differed in size (P-value = 0.016), concentration (P-value = 0.016) and protein content (P-value = 0.016). There was no significant difference in expressions of miR-19a-3p (P-value = 0.219) and miR-92a-3p (P-value = 0.094) between the two isolation kits. The new, adapted protocol described, enables simultaneous analysis of multiple elements when investigating potential biomarkers of CRC. |
| format | Article |
| id | doaj-art-8dc7b25ca1e04c22b0bd573b5fa2ec1c |
| institution | OA Journals |
| issn | 2045-2322 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-8dc7b25ca1e04c22b0bd573b5fa2ec1c2025-08-20T02:33:05ZengNature PortfolioScientific Reports2045-23222024-11-0114111010.1038/s41598-024-78497-xEstablishment of liquid biopsy procedure for the analysis of circulating cell free DNA, exosomes, RNA and proteins in colorectal cancer and adenoma patientsAndrea Čeri0Anita Somborac-Bačura1Marija Fabijanec2Andrea Hulina-Tomašković3Marko Matusina4Dijana Detel5Donatella Verbanac6Karmela Barišić7Department of Medical Biochemistry and Haematology, University of Zagreb Faculty of Pharmacy and BiochemistryDepartment of Medical Biochemistry and Haematology, University of Zagreb Faculty of Pharmacy and BiochemistryCentre for Applied Medical Biochemistry, University of Zagreb Faculty of Pharmacy and BiochemistryDepartment of Medical Biochemistry and Haematology, University of Zagreb Faculty of Pharmacy and BiochemistryDepartment of Medical Biochemistry and Haematology, University of Zagreb Faculty of Pharmacy and BiochemistryDepartment of Medical Chemistry, Biochemistry and Clinical Chemistry, University of Rijeka Faculty of MedicineDepartment of Medical Biochemistry and Haematology, University of Zagreb Faculty of Pharmacy and BiochemistryDepartment of Medical Biochemistry and Haematology, University of Zagreb Faculty of Pharmacy and BiochemistryAbstract Liquid biopsy has an underexplored diagnostic potential in colorectal cancer (CRC). Sufficient quantity and quality of its elements (circulating cell-free DNA (ccfDNA), exosomes and exosomal RNA) are essential for accurate results. The present study aims to establish the optimal protocol for handling liquid biopsy samples. Samples were obtained by collecting peripheral blood from colorectal adenoma patients in CellSave tubes. Plasma was separated within six hours using differential centrifugation and aliquots stored at – 20/– 80 °C until further processing. Three methods for isolation of ccfDNA, and two combinations of kits for isolation of exosomes and exosomal RNA were tested. The quality and quantity of ccfDNA isolates were evaluated. Exosomes were characterised by determining size, concentration, and total and specific protein content. Expression of chosen microRNAs, miR-19a-3p and miR-92-3p, which have been implicated in CRC progression, were determined. The vacuum-column-based kit showed the highest quantities of isolated ccfDNA (P-value < 0.001). Kits for exosome isolation significantly differed in size (P-value = 0.016), concentration (P-value = 0.016) and protein content (P-value = 0.016). There was no significant difference in expressions of miR-19a-3p (P-value = 0.219) and miR-92a-3p (P-value = 0.094) between the two isolation kits. The new, adapted protocol described, enables simultaneous analysis of multiple elements when investigating potential biomarkers of CRC.https://doi.org/10.1038/s41598-024-78497-xCirculating tumour DNAColorectal cancerExosomesLiquid biopsyMethodology challengesmicroRNA |
| spellingShingle | Andrea Čeri Anita Somborac-Bačura Marija Fabijanec Andrea Hulina-Tomašković Marko Matusina Dijana Detel Donatella Verbanac Karmela Barišić Establishment of liquid biopsy procedure for the analysis of circulating cell free DNA, exosomes, RNA and proteins in colorectal cancer and adenoma patients Scientific Reports Circulating tumour DNA Colorectal cancer Exosomes Liquid biopsy Methodology challenges microRNA |
| title | Establishment of liquid biopsy procedure for the analysis of circulating cell free DNA, exosomes, RNA and proteins in colorectal cancer and adenoma patients |
| title_full | Establishment of liquid biopsy procedure for the analysis of circulating cell free DNA, exosomes, RNA and proteins in colorectal cancer and adenoma patients |
| title_fullStr | Establishment of liquid biopsy procedure for the analysis of circulating cell free DNA, exosomes, RNA and proteins in colorectal cancer and adenoma patients |
| title_full_unstemmed | Establishment of liquid biopsy procedure for the analysis of circulating cell free DNA, exosomes, RNA and proteins in colorectal cancer and adenoma patients |
| title_short | Establishment of liquid biopsy procedure for the analysis of circulating cell free DNA, exosomes, RNA and proteins in colorectal cancer and adenoma patients |
| title_sort | establishment of liquid biopsy procedure for the analysis of circulating cell free dna exosomes rna and proteins in colorectal cancer and adenoma patients |
| topic | Circulating tumour DNA Colorectal cancer Exosomes Liquid biopsy Methodology challenges microRNA |
| url | https://doi.org/10.1038/s41598-024-78497-x |
| work_keys_str_mv | AT andreaceri establishmentofliquidbiopsyprocedurefortheanalysisofcirculatingcellfreednaexosomesrnaandproteinsincolorectalcancerandadenomapatients AT anitasomboracbacura establishmentofliquidbiopsyprocedurefortheanalysisofcirculatingcellfreednaexosomesrnaandproteinsincolorectalcancerandadenomapatients AT marijafabijanec establishmentofliquidbiopsyprocedurefortheanalysisofcirculatingcellfreednaexosomesrnaandproteinsincolorectalcancerandadenomapatients AT andreahulinatomaskovic establishmentofliquidbiopsyprocedurefortheanalysisofcirculatingcellfreednaexosomesrnaandproteinsincolorectalcancerandadenomapatients AT markomatusina establishmentofliquidbiopsyprocedurefortheanalysisofcirculatingcellfreednaexosomesrnaandproteinsincolorectalcancerandadenomapatients AT dijanadetel establishmentofliquidbiopsyprocedurefortheanalysisofcirculatingcellfreednaexosomesrnaandproteinsincolorectalcancerandadenomapatients AT donatellaverbanac establishmentofliquidbiopsyprocedurefortheanalysisofcirculatingcellfreednaexosomesrnaandproteinsincolorectalcancerandadenomapatients AT karmelabarisic establishmentofliquidbiopsyprocedurefortheanalysisofcirculatingcellfreednaexosomesrnaandproteinsincolorectalcancerandadenomapatients |