|1122-3||COMPARISON OF CT AND CY0 METHODS FOR QUANTITATIVE PCR (qPCR)|
|Autores:||João Vitor Dutra Molino (USP - Universidade de São Paulo) ; Tatiana Lopes (USP - Universidade de São Paulo) ; Daniela Araújo Viana Marques (USP - Universidade de São Paulo) ; Vivian Fonseca Gonzaga (USP - Universidade de São Paulo) ; Joas Lucas da Silva (USP - Universidade de São Paulo) ; André Moreni Lopes (USP - Universidade de São Paulo) ; Mario Hiroyuki Hirata (USP - Universidade de São Paulo) ; Priscila Gava Mazzola (UNICAMP - Universidade de Campinas) ; Maria Silvia Vicarri Gatti (UNICAMP - Universidade de Campinas) ; Adalberto Pessoa Júnior (USP - Universidade de São Paulo) |
The qPCR is a sophisticated and sensitive technique that involves, among the most common methods, the annealing of fluorescent flags (SybrGreen) or hybridization specific probes (TaqMan). Quantification of the amplified product is obtained by a comparison against a standard curve, constructed using different known concentrations of the target sequence, and correlates the intensity of fluorescence signals generated during the amplification cycles to the DNA concentration. This technique can be applied in diverse fields of microbiology, included in the biopharmaceutical industries which need an efficient procedure to control microorganism production.
Results of the qPCR were calculated using two different methods: Ct, which uses a threshold line defining a default level of fluorescence for all reactions; and the Cy0, a nonlinear fit of the function of Richard from the results of the qPCR reaction. However, when biological samples are used, inhibitors may be present and can change the efficiency of reactions. Due to its ability to ignore possible interferences in the results of the qPCR, the Cy0 was compared to the Ct method. Another factor evaluated was the accuracy of both methods, expressed as RSD (relative standard deviation), using central points as replicates. The RSD was evaluated for different parameters calculated with both methods.
The results indicate that the Cy0 method is more accurate for higher measurements of DNA. However, the parameters used were determined with relatively high concentrations of DNA. At low concentrations, little information about the maximum fluorescence is obtained, which makes the regression itself less accurate. No difference was observed on the quantification using both methods, showing no evidence of interference on the assays. However, it’s important to note the serial dilution method might be the responsible for the reduced interference observed, and the presence of interfering particles might not be discarded on less diluted samples.
In conclusion, both methods were equal since interference was not observed regarding measurement of the amplified product in qPCR. However, the accuracy of the Cy0 was higher in samples with higher concentration of DNA, while the Ct method is more accurate in the samples with reduced concentration.
Palavras-chave: quantitative PCR (qPCR), Ct method, Cy0 method, DNA quantification