Usporedba metoda za izolaciju RNA iz uzoraka egzosoma bolesnika s kolorektalnim karcinomom

master's thesis

• Title (english): Comparison of methods for the isolation of RNA from exosome samples of patients with colorectal carcinoma
• Author: Marko Borović
• Mentor: Andrea Hulina Tomašković (mentor)
• Committee member: Andrea Hulina Tomašković (predsjednik povjerenstva)
• Committee member: Anita Somborac Bačura (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: 2023-04-20, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Kolorektalni karcinom (CRC) predstavlja ozbiljan dijagnostički i terapijski problem na globalnoj razini zbog stalno rastuće
prevalencije. Multifaktorska je bolest, specifične etiologije za svakog bolesnika te je potrebna serija genskih mutacija tokom
dužeg razdoblja za razvoj invazivnog karcinoma. Egzosomi koji potječu iz stanica kolorektalnog karcinoma sadrže molekule
miRNA koje se povezuju s lošom prognozom tako što potiču proliferaciju, migraciju i invazivnost tumorskih stanica te
predstavljaju potencijalan biomarker pokazujući značajnu korelaciju s patološkim stanjem i progresijom kolorektalnog
karcinoma. Tekuća biopsija je neinvazivna metoda kojom se mogu izolirati cirkulirajući egzosomi i miRNA iz uzorka krvi što
može voditi velikom napretku u ranoj dijagnostici karcinoma kao i u njegovom praćenju. Cilj diplomskog rada je usporediti
metode Qiagen i ThermoFisher za izolaciju molekula RNA iz egzosoma kod bolesnika s kolorektalnim karcinomom. Početni
je uzorak puna krv iz čije se plazme izolira egzosomalna RNA metodom precipitacije i organske ekstrakcije. Koncentracija
dobivene RNA određuje se spektrofotmetrijski, dok se kvaliteta i integritet određuju metodom kapilarne elektroforeze.
Ekspresija referentnih gena B2M i PPIA određena je metodom Taqman qPCR prema kojoj se određuje relativna ekspresija
ciljnih gena. Također, uspoređuje se ekspresija ciljne miRNA (miR-19a-3p) za čiju se ekspresiju pretpostavlja da bi trebala
biti povišena kod bolesnika s kolorektalnim karcinomom te referentne miRNA miR-103a-3p u odnosu na „spike-in“ miRNA
UniSp6. Iz dobivenih se rezultata može zaključiti kako ne postoji statistički značajna razlika između metoda Qiagen i
ThermoFisher, odnosno metode su jednakovrijedne i može se koristiti bilo koja od dvije metode za određivanje ekspresije
miRNA bez značajne razlike u rezultatima. Unatoč niskoj koncentraciji izolirane RNA, uzorci su dovoljno kvalitetni za
određivanje ekspresije miRNA. Sukladno tome, potvrđuje se prisutnost referentne (miR-103a-3p) i ciljne (miR-19a-3p)
miRNA u svim uzorcima.

Abstract (english)

Colorectal cancer (CRC) represents a serious diagnostic and therapeutic problem at the global level due to its ever-increasing prevalence. It is a multifactorial disease, with a specific etiology for each patient, and a series of gene mutations over a longer period is required for the development of invasive cancer. Exosomes from colorectal cancer cells contain miRNA molecules that are associated with poor prognosis by promoting the proliferation, migration, and invasiveness of tumor cells. Furthermore, they represent a potential biomarker showing a significant correlation with the pathological state and progression of colorectal cancer. Liquid biopsy is a non-invasive method that can isolate circulating exosomes and miRNAs from a blood sample, which could lead to great progress in the early diagnosis of cancer as well as in its monitoring. The aim of this thesis is to compare the Qiagen and ThermoFisher methods for the isolation of RNA molecules from exosomes in patients with colorectal cancer. The initial sample is whole blood where exosomal RNA is isolated by precipitation and organic extraction from plasma exosomes. The concentration of the obtained RNA is determined spectrophotometrically, while the quality and integrity are determined by the capillary electrophoresis method. The expressions of reference genes
B2M and PPIA were determined by the Taqman qPCR method, which determines the relative expression of the target genes.
Additionally, the expression of the target miRNA (miR-19a-3p) is compared, as it is supposed to be elevated in patients with
colorectal cancer and reference miRNA miR-103a-3p relative to “spike-in” miRNA UniSp6. In conclusion, there was no
statistically significant difference between the Qiagen and ThermoFisher methods, therefore, the methods could be
considered equivalent and either of the two can be used to determine miRNA expression without a significant difference in
results. Despite the low concentration of isolated RNA, the samples are of sufficient quality to determine miRNA expression.
Accordingly, the presence of reference (miR-103a-3p) and target (miR-19a-3p) miRNA is confirmed within all samples.

• Keywords: kolorektalni karcinom; egzosomi; miRNA; Qiagen; ThermoFisher
• Keywords (english): colorectal carcinoma; exosomes; miRNA; Qiagen; ThermoFisher
• Language: croatian
• URN:NBN: urn:nbn:hr:163:683612
• Study programme: Title: Medical biochemistry; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra medicinske biokemije (magistar/magistra medicinske biokemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2023-05-08 13:10:53