Comparisons between RT-PCR, real-time PCR, and in vitro globin chain synthesis by α/β ratio calculation for diagnosis of α- from β-thalassemia carriers.

BACKGROUND Thalassemia, which may be due to point mutations, translocations, and deletions involving the α or βglobin gene, is the most prevalent single gene disorder in Iran.This study aims to calculate the α/β ratio in normal cases, α- and β-thalassemia carriers by RT-PCR, real-time PCR, and in vitro globin chain synthesis (GCS) in order to establish the most accurate technique to distinguish between α- and β-thalassemia carriers in suspicious cases. METHODS The α/β ratios were calculated in all samples by RT-PCR, real-time RT-PCR, and in vitro GCS. RESULTS Using RT-PCR, the ratios were 1.09 ± 0.07 in normal samples, 1.2 ± 0.17 in β-thalassemia, 1.08 ± 0.19 in mild α-thalassemia, and 0.96 ± 0.19 in severe α-thalassemia carriers. In real-time RT-PCR, the ratios were 2.21 ± 1.36 in normal samples, 5.12 ± 1.83 in β-thalassemia, 2.88 ± 0.81 in mild α-thalassemia, and 1.18 ± 0.52 in severe α-thalassemia carriers. With GCS, the ratios were 1.03 ± 0.1 in normal samples, 1.9 ± 0.37 in β-thalassemia, 0.8 ± 0.13 in mild α-thalassemia, and 0.59 ± 0.12 in severe α-thalassemia carriers. CONCLUSION To determine the most accurate technique, we statistically analyzed the α/β ratios obtained from the three standard methods. The ratio obtained by GCS and real-time PCR were helpful in distinguishing between α and β carriers in suspicious patients in whom the mutation detection was limited and the risk for offspring was not clear. The use of this technique is more obvious when time is restricted (i.e. during the pregnancy period).