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

Ermenc, Teja

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Title:	Optimizacija procesiranja vzorcev sline pri raku glave in vratu za študije bioloških označevalcev
Authors:	ID Ermenc, Teja (Author) ID Potočnik, Uroš (Mentor) More about this mentor... ID Čelešnik, Helena Sabina (Comentor)
Files:	UN_Ermenc_Teja_2018.pdf (1,71 MB) MD5: CFC34B6C566C344A15384910AB0D38B2 PID: 20.500.12556/dkum/80ccd20d-d0eb-4a8a-88c8-55f0b83b2be3
Language:	Slovenian
Work type:	Bachelor thesis/paper
Typology:	2.11 - Undergraduate Thesis
Organization:	FKKT - Faculty of Chemistry and Chemical Engineering
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, izolacija iz sline, metilacija
Place of publishing:	Maribor
Publisher:	[T. Ermenc]
Year of publishing:	2018
PID:	20.500.12556/DKUM-71832
UDC:	601.4:577.213.3/.7(043.2)
COBISS.SI-ID:	21977110
NUK URN:	URN:SI:UM:DK:DEHELEE4
Publication date in DKUM:	05.10.2018
Views:	1639
Downloads:	189
Metadata:
Categories:	KTFMB - FKKT
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Licences

License:	CC BY 4.0, Creative Commons Attribution 4.0 International

Link:	http://creativecommons.org/licenses/by/4.0/
Description:	This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.
Licensing start date:	31.08.2018

Secondary language

Language:	English
Title:	Optimization of saliva sample processing for head and neck cancer biomarkers studies
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.
Keywords:	head and neck cell carcioma cancer, biomarkers, isolation from saliva, methylation

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