Usporedba metoda izdvajanja slobodne cirkulirajuće DNA iz uzoraka tekuće biopsije pacijenata s kolorektalnim adenomom

master's thesis

• Title (english): Comparison of methods for isolating circulating cell-free DNA from liquid biopsy samples of patients with colorectal adenoma
• Author: Marko Matusina
• Mentor: Andrea Čeri (mentor)
• Committee member: Andrea Čeri (predsjednik povjerenstva)
• Committee member: Marija Grdić Rajković (član povjerenstva)
• Committee member: Lidija Bach-Rojecky (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2024-11-06, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Kolorektalni karcinom (CRC) jedan je od vodećih uzroka smrtnosti povezanih s malignim bolestima u svijetu, pri
čemu rana dijagnostika predstavlja temeljni preduvjet za uspješno liječenje. Slobodna cirkulirajuća DNA (ccfDNA) je
dvolančana, fragmentirana DNA prisutna izvan stanica, koja se može detektirati i analizirati iz uzoraka tekuće
biopsije. Kod pacijenata s malignim oboljenjima, dio ccfDNA čini cirkulirajuća tumorska DNA, čija koncentracija i
specifične molekularne promjene odražavaju dinamiku bolesti, omogućujući primjenu analize ccfDNA kao
dijagnostičkoga, prognostičkoga i prediktivnoga biljega. S obzirom na nisku koncentraciju i izraženu fragmentiranost
ccfDNA u biološkim uzorcima, ključno je uspostaviti optimalnu metodu izdvajanja te provesti temeljitu
standardizaciju i validaciju svih postupaka tekuće biopsije. Cilj ovoga rada je usporediti tri komercijalno dostupne
metode za izdvajanje ccfDNA: NucleoSpin cfDNA XS Kit (Machery Nagel, Njemačka), QIAamp ccfDNA/RNA Kit
(Qiagen, Njemačka) i QIAamp Circulating Nucleic Acid Kit (Qiagen). Ispitivane metode izdvajanja temelje se na
selektivnoj adsorpciji ccfDNA na kolone od silikatnih čestica. Koncentracija izdvojene ccfDNA u svakom izolatu
određena je fluorimetrijskim mjerenjem, dok su kvaliteta i integritet ocijenjeni detekcijom fragmenata pomoću
automatizirane mikrovolumne gel-elektroforeze u kapilari na čipu. Metodom QIAamp Circulating Nucleic Acid Kit
dobivene su najveće koncentracije ccfDNA i jedina je od ispitivanih metoda kojom je u svim uzorcima detektiran
elektroforetski vršak koji odgovara ccfDNA, čime se ističe kao optimalna za daljnju primjenu.

Abstract (english)

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide, and early diagnosis is a
crucial prerequisite for successful treatment. Circulating cell-free DNA (ccfDNA) is double-stranded, fragmented
DNA found outside cells, detectable and analysable from liquid biopsy samples. In patients with malignant diseases, a
portion of ccfDNA corresponds to circulating tumor DNA, whose concentration and specific molecular changes reflect
disease dynamics, allowing ccfDNA to serve as a diagnostic, prognostic, and predictive biomarker. Given the low
concentration and significant fragmentation of ccfDNA in biological samples, establishing an optimal isolation method
is essential, alongside thorough standardization and validation of all liquid biopsy procedures. This study compares
three commercially available methods for isolating ccfDNA: NucleoSpin cfDNA XS Kit (Machery Nagel, Germany),
QIAamp ccfDNA/RNA Kit (Qiagen, Germany), and QIAamp Circulating Nucleic Acid Kit (Qiagen). The extraction
methods examined here rely on the selective adsorption of ccfDNA to silica-based columns. The concentration of
isolated ccfDNA was determined by fluorimetric measurment, while quality and integrity were assessed by detecting
fragments using automated microvolume gel-electrophoresis on a chip. The QIAamp Circulating Nucleic Acid Kit
yielded the highest ccfDNA concentrations and was the only method among those tested to detect an electrophoretic
peak corresponding to ccfDNA in all samples, highlighting it as optimal for further application.

• Keywords: kolorektalni karcinom; kolorektalni adenom; tekuća biopsija; slobodna cirkulirajuća DNA; izdvajanje DNA; usporedba metoda
• Keywords (english): colorectal cancer; colorectal adenoma; liquid biopsy; circulating cell-free DNA; DNA extraction; method comparison
• Language: croatian
• URN:NBN: urn:nbn:hr:163:747237
• Study programme: Title: Medical biochemistry - integrated undergraduate and graduate - university study; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: sveučilišni magistar medicinske biokemije i laboratorijske medicine (sveučilišni magistar medicinske biokemije i laboratorijske medicine)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2025-01-29 12:04:14