Odabir i evaluacija kandidatnih referentnih gena za analizu genske ekspresije u mozgu gušterica roda Podarcis

master's thesis

• Title (english): Selection and evaluation of candidate reference genes for gene expression analysis in the brain of Podarcis lizards
• Author: Ana-Marija Petrović
• Mentor: Sofia Ana Blažević (mentor)
• Committee member: Sofia Ana Blažević (predsjednik povjerenstva)
• Committee member: Maja Matulić (član povjerenstva)
• Committee member: Inga Urlić (član povjerenstva)
• Committee member: Martina Šeruga Musić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2023-09-27, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Agresivnost je oblik kompetitivnog ponašanja koji snažno utječe na odnose između jedinki i
vrsta. Izraženo agresivno ponašanje je primijećeno u odnosima jedinki vrsta Podarcis sicula
(primorska gušterica) i Podarcis melisellensis (krška gušterica) na otocima i kontinentalnim
dijelovima jadranske obale, gdje agresivnija P. sicula potiskuje P. melisellensis i smanjuje njen
areal. Poznata je uloga monoamina u inicijaciji agresivnog ponašanja, naročito dopamina,
serotonina i noradrenalina. Razlike u njihovim koncentracijama su u osnovi brojnih ponašanja i
odraz su diferencijalne ekspresije njihovih metaboličkih gena. Profiliranje ekspresije gena
pomoću RT-qPCR je robusna tehnika koja se sve češće koristi u neuroetološkim istraživanjima.
Preduvjet za RT-qPCR analize je određivanje i provjera optimalnih referentnih gena koji će
služiti za normalizaciju dobivenih rezultata. Cilj ovog diplomskog rada je analizirati stabilnost
ekspresije deset potencijalnih referentnih gena (ACTB, GAPDH, GUSB, HMBS, HPRT1, PGK1,
SDHA, TBP, TFRC i YWHA) i odabrati pet za buduće analize genske ekspresije, kao i
optimizirati temperature taljenja odabranih parova početnica i učinkovitost umnažanja ciljanih
sekvenci. Kao najstabilniji referentni geni su se pokazali: PGK1, YWHAZ, HMBS, HPRT1 i
GUSB.

Abstract (english)

Aggression is a form of competitive behavior that strongly affects relationships between
individuals and species. Pronounced aggressive behavior was observed in the relationship
between the species Podarcis sicula (coastal lizard) i Podarcis melisellensis (karst lizard) on the
islands and continental parts of the Adriatic coast, where the more aggressive P. sicula
suppresses P. melisellensis and reduces its areal. The role of monoamines in the initiation of
aggressive behavior is already known; dopamine, serotonin and noradrenaline in particular. The
difference in their amounts is the basis of numerous types of behaviors and is a reflection of the
differential expression of their metabolic genes. Gene expression profiling using RT-qPCR is a
robust technique that is increasingly being used in neuroethological research. A prerequisite for
RT-qPCR analysis is the determination and validation of optimal reference genes that will serve
to normalize the obtained data. The aim of this thesis is to analyze the expression stability of ten
potential reference genes (ACTB, GAPDH, GUSB, HMBS, HPRT1, PGK1, SDHA, TBP, TFRC
and YWHA) and to select five for future gene expression analysis, as well as to optimize the
melting temperatures of the selected primer pairs and amplification efficiencies of target
sequences. The most stably expressed reference genes were determined to be: PGK1, YWHAZ,
HMBS, HPRT1 and GUSB.

• Keywords: smjernice MIQE; referentni geni; reproducibilnost; RT-qPCR
• Keywords (english): MIQE guidelines; reference genes; reproducibility; RT-qPCR
• Language: croatian
• URN:NBN: urn:nbn:hr:217:419515
• Project: Number: UIP-2019-04-8469; Title: Dopaminska regulacija kompetitivnog ponašanja u kohabituirajućim populacijama guštera Podarcis siculus i Podarcis melisellensis; Title: Dopamine regulation of competitive behavior in coexisting populations of lizards Podarcis siculus and Podarcis melisellensis; Acronym: BOLDer; Leader: Sofia Ana Blažević; Jurisdiction: Croatia; Funder: HRZZ; Funding stream: UIP
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2025-09-27
• Created on: 2023-10-17 11:17:57