20.500.12556/RUL-17717 Comparison of methods for relative quantification of gene expression using real-time PCR 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. 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. molecular genetics genes gene expression quantitative real-time PCR methods molekularna genetika geni gensko izražanje kvantitativni PCR v realnem času metode true false false Angleški jezik Slovenski jezik Delo ni kategorizirano 2014-07-11 13:54:52 2014-07-11 13:54:52 2022-05-20 03:45:00 0000-00-00 00:00:00 2012 0 0 Str. 97-106 št. 2 Letn. 100 2012 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 575 1581-9175 3162504 http://aas.bf.uni-lj.si/zootehnika/100-2012/PDF/100-2012-2-97-106.pdf 1 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=17717 Biotehniška fakulteta Acta agriculturae Slovenica 0 0 0