Comparison of methods for relative quantification of gene expression using real-time PCR

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

Quantitative real-time PCR (qPCR) has become a widely used tool for quantifying gene expression. Several methods for relative quantification have been developed, enabling rapid and reliable detection and quantification of specific nucleic acids. These methods, based on qPCR include: the standard curve method, the efficiency calibrated method and the 2[-deltadelta]Cq method. Here we analyzed if these three methods generate comparable results. To evaluate their performance, we analyzed the expression of the nuclease gene MS53_0284 from Mycoplasma synoviae type strain WVU 1853 during in vitro infection of CEC-32 cells, using qPCR. As determined, all three methods generated comparable and reliable results when all necessary conditions were fulfiled. Also, the efficiency calibrated and the standard curve methods were more suitable for quantifying small differences in relative gene expression than the 2[-deltadelta]Cq method.

• Language: English
• Keywords: molecular genetics, genes, gene expression, quantitative real-time PCR, methods
• Work type: Not categorized
• Typology: 1.01 - Original Scientific Article
• Organization: BF - Biotechnical Faculty
• Year: 2012
• Number of pages: Str. 97-106
• Numbering: Letn. 100, št. 2
• PID: 20.500.12556/RUL-17717
• UDC: 575
• ISSN on article: 1581-9175
• COBISS.SI-ID: 3162504
• 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

Metoda kvantitativne verižne reakcije s polimerazo v realnem času (qPCR) je postala najpogosteje uporabljen način analize izražanja genov. Razvitih je bilo več metod za relativno kvantifikacijo genske ekspresije, ki omogočajo hitro in zanesljivo detekcijo ter kvantifikacijo specifičnih nukleinskih kislin. Mednje spadajo: metoda z umeritveno krivuljo, metoda z upoštevanjem učinkovitosti pomnoževanja in metoda po enačbi 2[-deltadelta]Cq. V tej študiji smo z analizirajem izražanja gena MS53_0284, ki kodira nukleazo pri bakteriji Mycoplasma synoviae WVU 1853, po okužbi celic CEC-32 in vitro s qPCR preverili primerljivost rezultatov, dobljenih z uporabo omenjenih metod. Pokazali smo, da z upoštevanjem potrebnih pogojev z omenjenimi metodami pridobimo primerljive rezultate ter da sta metoda z umeritveno krivuljo in metoda z upoštevanjem učinkovitosti pomnoževanja primernejši za ugotavljanje majhnih razlik v izražanju genov kot metoda po enačbi 2[-deltadelta]Cq.

• Keywords: molekularna genetika, geni, gensko izražanje, kvantitativni PCR v realnem času, metode