Identifikacija optimalne kombinacije referenčnih genov v primarnih človeških perinatalnih mezenhimskih matičnih/stromalnih celicah za kvantitativno verižno reakcijo s polimerazo

Suša, Hana (avtor), Zupan, Janja (mentor), Trojner Bregar, Andreja (komentor)

Perinatalna tkiva so dostopen vir mezenhimskih matičnih/stromalnih celic (MSC) z velikim raziskovalnim in terapevtskim potencialom. Za zanesljivo vrednotenje izražanja genov v teh celicah je potrebna ustrezna normalizacija rezultatov. Ta temelji na uporabi referenčnih genov, katerih izražanje mora biti v proučevanih celicah in izbranih eksperimentalnih pogojih čim bolj stabilno. Namen magistrskega dela je bil med šestimi kandidatnimi referenčnimi geni določiti najprimernejšo kombinacijo za normalizacijo izražanja genov v nediferenciranih primarnih humanih MSC iz posteljice (PL) in amnijske membrane (AM). Celokupno ribonukleinsko kislino (RNA) smo izolirali iz šestnajstih vzorcev, pridobljenih od dvanajstih preiskovank, ter določili njeno koncentracijo in čistost. En vzorec smo zaradi prenizke koncentracije RNA izključili iz nadaljnje analize. Izražanje kandidatnih referenčnih genov smo določili s kvantitativno verižno reakcijo s polimerazo v realnem času (qPCR). Zanesljivost meritev smo ovrednotili na podlagi učinkovitosti in specifičnosti pomnoževanja ter ponovljivosti tehničnih ponovitev, stabilnost izražanja pa z algoritmom geNorm. Izražanje vseh šestih kandidatnih genov je bilo merljivo in dobro ponovljivo. Pet genov je izkazalo ustrezno učinkovitost pomnoževanja, medtem ko je ta pri genu za beta-glukuronidazo (GUSB) znašala 80,3 odstotka, zaradi česar ga nismo vključili v nadaljnjo analizo stabilnosti izražanja kandidatnih referenčnih genov. Kot najstabilnejša se je izkazala kombinacija gena za gliceraldehid-3-fosfat dehidrogenazo (GAPDH) in gena za beta-aktin (ACTB). Analiza parne variacije je pokazala, da za zanesljivo normalizacijo zadoščata dva referenčna gena. Na podlagi rezultatov lahko sklenemo, da je par GAPDH–ACTB najprimernejša kombinacija med petimi geni, vključenimi v končno analizo, in za skupno analizirani nabor 15 vzorcev PL- in AM-MSC. O stabilnosti izražanja GUSB na podlagi te analize ne moremo sklepati, pred njegovo morebitno ponovno vključitvijo pa bi bilo treba optimizirati qPCR test. Izbrani par bi bilo treba pred rutinsko uporabo potrditi na večjem in neodvisnem naboru vzorcev.

• Jezik: Slovenski jezik
• Ključne besede: perinatalne mezenhimske matične/ stromalne celice (MSC), referenčni geni, normalizacija izražanja genov, kvantitativna verižna reakcija s polimerazo v realnem času (qPCR), stabilnost izražanja genov
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FFA - Fakulteta za farmacijo
• Založnik: [H. Suša]
• Leto izida: 2026
• PID: 20.500.12556/RUL-189073
• UDK: 577.218(043.2)
• COBISS.SI-ID: 293955843
• Datum objave v RUL: 01.10.2026

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Identification of an optimal reference gene combination in primary human perinatal mesenchymal stem/stromal cells for quantitative polymerase chain

Izvleček

Perinatal tissues are an accessible source of mesenchymal stem/stromal cells (MSCs) with considerable research and therapeutic potential. Reliable evaluation of gene expressions in these cells requires appropriate normalization of the results. This process is based on reference genes whose expression should remain as stable as possible in the investigated cells and under the selected experimental conditions. The aim of this master’s thesis was to identify the most suitable combination of six candidate reference genes for normalizing gene expression in undifferentiated primary human MSCs derived from the placenta and amniotic membrane. Total ribonucleic acid (RNA) was isolated from sixteen samples obtained from twelve participants, and its concentration and purity were determined. One sample was excluded from further analysis because of insufficient concentration. Candidate reference gene expression was measured using real-time quantitative polymerase chain reaction (qPCR). Measurement reliability was evaluated based on amplification efficiency and specificity as well as the reproducibility of technical replicates, while expression stability was assessed using the geNorm algorithm. The expression of all six candidate genes was measurable and showed good reproducibility. Five genes exhibited acceptable amplification efficiency, whereas the amplification efficiency of the gene encoding beta-glucuronidase (GUSB) was 80.3%. Consequently, GUSB was excluded from the subsequent expression stability analysis. The combination of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the gene encoding beta-actin (ACTB) was identified as the most stable pair. Pairwise variation analysis indicated that two reference genes were sufficient for reliable normalization. Based on these results, the GAPDH–ACTB pair can be considered the most suitable combination among the five genes included in the final analysis and for the combined set of 15 placenta- and amniotic membrane-derived MSC samples. No conclusion regarding the expression stability of GUSB can be drawn from this analysis, and its qPCR assay should be optimized before the gene is reconsidered for inclusion. The selected pair should be validated in a larger and independent sample set before routine use.

• Ključne besede: perinatal mesenchymal stem/stromal cells (MSCs), reference genes, gene expression normalization, real-time quantitative polymerase chain reaction (qPCR), gene expression stability