Optimizacija procesiranja vzorcev sline pri raku glave in vratu za študije bioloških označevalcev

diplomsko delo univerzitetnega študijskega programa I. stopnje

Teja Ermenc (Author), Uroš Potočnik (Mentor), Helena Sabina Čelešnik (Co-mentor)

Abstract

Namen diplomske naloge je bil optimizirati pridobivanje genetskega materiala iz vzorcev sline pri bolnikih z rakom glave in vratu za analizo bioloških označevalcev. Ustna tekočina oz. slina vsebuje genetski material, celice in različne molekule, ki lahko odražajo bolezenske spremembe. Zaradi tega bi lahko ustna tekočina služila kot stroškovno učinkovita in manj invazivna alternativa biopsiji za zgodnje odkrivanje raka. Za izolacijo nukleinskih kislin in proteinov iz krvi obstaja na Centru za humano genomiko in farmakogenomiko Medicinske fakultete v Mariboru že ustaljen postopek, za izolacijo iz sline pa ta še ni bil vzpostavljen. V nalogi smo želeli pripraviti laboratorijske protokole za učinkovito pridobivanje nukleinskih kislin in proteinov iz izpirkov ustne votline s TRI-reagentom. V ta namen smo na vzorcih moje sline testirali različne izolacijske pogoje, kot so različna količina sline, različne količine reagentov, izolacija iz ustnega izpirka ali iz peleta ustnega izpirka. Ugotovili smo, da je iz peletiranega izpirka možno izolirati tako DNA kot RNA, vendar je RNA slabše kvalitete in vsebuje precej razgradnih produktov, najverjetneje zaradi delovanja ribonukleaz v slini. Vzpostavljen postopek izolacije smo nato uporabili na vzorcih izpirkov ustne votline bolnikov z rakom glave in vratu. Po pričakovanjih so izolati DNA in RNA kazali precejšnjo stopnjo razgradnje, saj so vzorci pred zamrznjenjem nekaj časa čakali na sobni temperaturi v zdravniški ordinaciji. Izolati DNA so bili dovolj kvalitetni za genotipizacijo in metilacijske analize, integriteta RNA pa je bila prenizka za analize genske ekspresije. Predvidevamo, da bi z odvzemanjem izpirkov neposredno v stabilizacijsko raztopino lahko izboljšali kvaliteto izolatov.

Keywords

rak glave in vratu;biološki označevalci;optimizacija iz sline;metilacija;diplomske naloge;

Data

Language:	Slovenian
Year of publishing:	2018
Typology:	2.11 - Undergraduate Thesis
Organization:	UM FKKT - Faculty of Chemistry and Chemical Engineering
Publisher:	[T. Ermenc]
UDC:	601.4:577.213.3/.7(043.2)
COBISS:	21977110
Views:	712
Downloads:	130
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Optimization of saliva sample processing for head and neck cancer biomarkers studies
Secondary abstract:	The purpose of this work was to optimize the isolation procedure of genetic material from the saliva samples of patients with head and neck cancer for the analyses of biomarkers. Oral fluid (i.e. saliva) contains genetic material, cells and various molecules that can reflect disease changes. Oral fluid could therefore serve as a cost-effective and noninvasive choice of biological sample for early cancer detection. For the isolation of nucleic acids and proteins from blood samples, the Center for Human Molecular Genetics and Pharmacogenomics at the Faculty of Medicine in Maribor follows an established procedure. However, no protocol has so far been established for isolations from saliva. In this work, we set out to determine the laboratory protocols for efficient extraction of nucleic acids and proteins from oral cavity rinses by using the TRI-reagent. To that end, we tested different isolation conditions such as varying amounts of oral samples, different quantities of reagents, nucleic acid extraction directly from oral rinses or from pelleted oral rinses. We found out that both DNA and RNA can be isolated from pellets of oral rinses, but RNA appeared of poor quality and contained a lot of degradation products, most likely due to the action of ribonucleases in the saliva. Following procedure optimization, we applied the established protocol to the samples derived from patients with head and neck cancer. Unsurprisingly, DNA and RNA isolates exhibited a significant degree of degradation, likely due to the time delay between saliva acquisition and its freezing. The samples were waiting at room temperature in the doctor's office for some time before being frozen. Nonetheless, DNA isolates were of sufficient quality for genotyping and methylation analyses. The integrity of RNA, on the other hand, was too low for gene expression analyses. We presume that collecting the oral rinses directly into a stabilization solution could improve the quality of the isolates.
Secondary keywords:	head and neck cancer;biomarkers;optimization from saliva;methylation;
URN:	URN:SI:UM:
Type (COBISS):	Bachelor thesis/paper
Thesis comment:	Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Pages:	IX, 36 str.
ID:	10956064