Analysis of [beta]/[alpha] Globin Ratio by Using Relative qRT-PCR for Diagnosis of Beta-Thalassemia Carriers

Background Current routine tests for premarital screening of [beta]-thalassemia carriers are not applicable for diagnosis of rare atypical minor [beta]-thalassemia cases. A more specialized laboratory evaluation for them is the measurement of [beta]/[alpha] chain synthesis ratio with the assistance of radioactive amino acids. This method is also no longer routinely accessible. Consequently it is required to establish a rapid, trouble-free, and reliable method that encompasses all the cases of [beta]-thalassemia carriers. Therefore we have determined [beta]/[alpha]-globin mRNA ratio by applying relative qRT-PCR in various [beta]-thalassemia patients. Methods Reticulocytes RNA extraction and subsequent cDNA synthesis were performed, followed by relative qRT-PCR for [alpha]- and [beta]-globin chain genes and [beta]-actin gene as an endogenous reference. [beta] / [alpha]-Globin gene ratio was then evaluated with the Pfaffl method. Results The mean of [beta]/[alpha] ratio was 0.99, 0.81, 0.69, and 0.69 for normal population, minor, intermediate, and major [beta]-thalassemia, respectively. Approximately 6% of cases with minor thalassemia RBC index and normal HbA2 and having a decreased [beta]/[alpha] ratio were located in the minor [beta]-thalassemia group. The mean of [beta]/[alpha] mRNA ratio in normal individuals and minor [beta]-thalassemia was significantly different with all other groups (P-value < 0.05). Nevertheless, there was no such association between [beta]/[alpha] mRNA ratio in major and intermediate [beta]-thalassemia. Conclusion According to the significant differences achieved, no overlapping between minor [beta]-thalassemia and normal group, capability of diagnosing atypical minor [beta]-thalassemia, and accessibility of this technique, we can declare that this method could be suggested as a routine premarital screening test for [beta]-thalassemia carriers. [PUBLICATION ABSTRACT]