PCR w czasie rzeczywistym. Metody analizy danych Jaros³aw Tyburski 1 , Anna Studziñska 1 , Patrycja Daca 2 , Andrzej Tretyn 1 1 Zak³ad Biotechnologii, Instytut Biologii Ogólnej i Molekularnej, Uniwersytet Miko³aja Kopernika, Toruñ 2 Zak³ad Medycyny S¹dowej, Collegium Medicum w Bydgoszczy, Uniwersytet Miko³aja Kopernika, Toruñ. PCR in real time. The methods of data analysis Summary The accuracy of real-time PCR (RT-PCR) experiment is strongly dependent on the mathematical models of data analysis, on which the quantitative meth- ods are based. In this review, we discuss the key steps of analysing data from real-time PCR experiments. These are the treshold cycle determination, estima- tion of real-time PCR amplification efficiency and amplicon quantification. The fit point method and the second derivative method are commonly used to de- termine a treshold cycle value, which is a cycle number in the early exponential phase of PCR that is used to calculate the initial amount of template DNA. The amplification efficiency calculation is usually based on the data collected from a standard curve. However, in the alternative methods, the amplification effi- ciency of an individual reaction is calculated from the kinetics of the reaction. Quantification of amplicon levels can be either absolute or relative. In absolute quantification method, the initial concentration of target template in un- knowns, based on their cycle treshold values, and the construction of an abso- lute standard curve for each individual amplicon is required. Relative quantifica- tion can be done by the use of the relative standard curve method or the com- parative method. In the first one, the initial amount of unknown samples is cal- culated from the standard curve of specific gene and normalized to the input amount of a reference gene which is also calculated from the standard curve. The comparative method is a mathematical model that is based on the differ- ences in the normalized amplicon levels between the unknowns and control sample. There are also models that combine gene quantification and normaliza- tion into a single calculation. Key words: Real-time PCR, methods of detection the treshold cycle, efficiency of PCR, quantification in real-time PCR. PRACE PRZEGL¥DOWE Adres do korespondencji Jaros³aw Tyburski, Zak³ad Biotechnologii, Wydzia³ Biologii i Nauk o Ziemi, Instytut Biologii Ogólnej i Molekularnej, Uniwersytet Miko³aja Kopernika, ul. Gagarina 9, 87-100 Toruñ; e-mail: tybr@uni.torun.pl 1 (80) 86–96 2008