Comparisons between RT-PCR, Real-time PCR, and In Vitro Globin Chain Synthesis by a/b Ratio Calculation for Diagnosis of a- from b-thalassemia Carriers

T halassemia syndromes are the most prevalent autosomal recessive single gene disorders in Iran.1,2 Interactions of different types of hemoglobinopathies can lead to thalassemia syndromes with a variety of phenotypes that range from asymptomatic to severe anemia. The diversity of thalassemia phenotypes depends on the amount of imbalances created between and non-globin chains.3,4 In normal erythropoiesis, there is a subtle balance between the productions of and -globin chains. Ineffective erythropoiesis, decreased erythrocyte lifespan, and clinical manifestations of -thalassemia are all related to the extent of imbalances in the globin chain. The defective -globin gene is responsible for the production of excessive -globin chains, which would be precipitated in red blood cells (RBCs) and would lead to ineffective erythropoiesis.5,6 The most direct method for evaluating the imbalances between and -globin chain ratios is in vitro globin chain synthesis (GCS).3,4 Recently, Irenge and Chaisue4,7,8 have developed a quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) that quanti es the expression of the defective -globin gene, which has revealed that qRT-PCR is an accurate technique to evaluate the transcriptional impact of the -globin gene. Chaisue et al.4 also measured the / ratio by the real-time PCR (qRT-PCR) and 2CT method and determined the presence of a relationship between the / ratio and disease severity. According to their ndings, the / ratio in -thalassemia carriers was less than non-carriers, whereas in -thalassemia carriers, this ratio was more when compared with non-carriers. By following the methods used by Han et al.,9 it has been shown that the / and /( + )-globin gene mRNA ratios varied signi cantly among normal adults, normal infants, -thalassemia minors, and -thalassemia majors. According to reports, real-time PCR (qRTPCR) compared with competitive RT-PCR was proven to be faster, less labor-intensive, and did not need molecular carryover.4,10 Khatami et al.11 measured the / ratio using GCS to differentiate rare types of normal A2 heterozygous -thalassemia from -thalassemia. According to their results, this method was capable of differentiating between silent -thalassemia and -thalassemia carriers. In the present study we determined the / globin and their mRNA ratios using GCS, RT-PCR, and qRT-PCR.