Setting up a gene expression study for tissue cells by method of quantitative real-time PCR

Dušanić, Daliborka (author), Bolha, Luka (author), Narat, Mojca (author), Oven, Irena (author)

Gene expression analysis by quantitative reverse transcription polymerase chain reaction (RT-qPCR) allows accurate and sensitive measurment of gene expression levels. However, a series of steps needs to be taken to ensure the relevance, accuracy, correct interpretation and repeatability of the RT-qPCR experiment. We describe here a simple experiment of determining relative gene expression for caspase 8 gene in chicken chondrocytes treated with an apoptosis inducing compound 5-fluorouracil. We use this example to point out some important guidelines in setting up a gene expression study in tissue cells, analyzing and interpreting the results and reporting on the findings.

• Language: English
• Keywords: molecular genetics, RT-qPCR, gene expression, tissue cells
• Work type: Not categorized
• Typology: 1.01 - Original Scientific Article
• Organization: BF - Biotechnical Faculty
• Year: 2012
• Number of pages: Str. 19-28
• Numbering: Letn. 100, št. 1
• PID: 20.500.12556/RUL-17710
• UDC: 575
• ISSN on article: 1581-9175
• COBISS.SI-ID: 3081864
• Publication date in RUL: 11.07.2014

Record is a part of a journal

• Title: Acta agriculturae Slovenica
• Shortened title: Acta agric. Slov.
• Publisher: Biotehniška fakulteta
• ISSN: 1581-9175
• COBISS.SI-ID: 213840640

Secondary language

• Language: Slovenian

Abstract

Analiza izražanja genov z metodo reverznega pomnoževanja in kvantitativne verižne reakcije s polimerazo v realnem času (RT-qPCR) omogoča natančno in občutljivo določanje nivoja izražanja tarčnih genov. Vendar je za zagotavljanje točnosti, natančne interpretacije in ponovljivosti eksperimenta, predvsem pa relevantnosti rezultatov, pomembno natančno poznavanje korakov postopka. V tem prispevku opisujemo enostaven poskus, v katerem smo v celični liniji kokošjih hondrocitov, ki smo jih tretirali s spojino 5-fluorouracil, ki sproži apoptozo, določili relativno izražanje gena, ki kodira encim kaspazo 8.Ta primer smo uporabili za izpostavitev nekaterih pomembnih smernic pri oblikovanju študije izražanja genov v tkivnih celicah, analizi in interpretaciji rezultatov ter oblikovanju zaključkov.

• Keywords: molekularna genetika, kvantitativni PCR v realnem času, genetsko izražanje, tkivne celice