control (Jain et al., 2006). The sequences of the primers used were
listed in Table 1. The PCR products were verified via a melting curve
analysis by changing the temperature from 60 C to 95 C to
monitor dissociation of double strand DNA and by running an
agarose gel electrophoresis. Amplification efficiencies (E) of the
primers were determined using the standard curve method and
calculated according to the equation E (%) ¼ (101/slope 1) 100.
All primer sets showed E values of more than 90%.
2.5. Statistical analysis
The collected data were subjected to one-way analysis of variance
(ANOVA) using the SPSS statistics package, version 21 (IBM
Inc., USA), and the means (n ¼ 4) were separated using Duncan's
multiple range test at p < 0.05.