[Quantitative analysis of human globin gene expression in beta-thalassemia using real-time RT-PCR]

Whole blood samples were collected from 100 normal healthy adults, from umbilical cord of 33 newborn infants, 111 individuals with beta-thalassemia minor (beta(T)/beta(A),alphaalpha/alphaalpha) and 39 with beta-thalassemia major (beta(T)/beta(T),alphaalpha/alphaalpha). Prior to quantitative analysis of globin gene expression, DNA was extracted from all blood samples and used for beta-thalassemia genotype analysis. Different types of beta globin gene mutations were analyzed using reverse dot blotting (RDB) method. Total RNA were extracted and subjected to real-time RT-PCR for quantitative measurement of alpha, beta and gamma globin mRNA using three sets of primers and fluorescent-labeled probes, designed according to the sequences of alpha, beta and gamma human globin gene. Real-time RT-PCR was performed in ABI 7700 system. Following the real-time RT-PCR, the mean values of alpha, beta and gamma globin mRNA were calculated and the ratios of alpha/beta, alpha/(beta + gamma ) and gamma /(beta + gamma ) were determined to characterize the relative expression levels of different globin genes among normal adult, infant, beta-thalassemia minor and beta-thalassemia major patients. The resultant data were analyzed using SPSS 10.0 software to determine statistical significance of human globin gene expression among normal controls and beta-thalassemia patients. Due to vast variations of the mean globin gene mRNA levels among different groups, log conversion of alpha/beta + 1, alpha/(beta + gamma ) + 1 and gamma /(beta + gamma ) +1 was used for statistical analyses and intergroup comparison. The alpha/beta globin gene mRNA ratios were determined to be 4.62+/-1.20, 7.81+/-2.89, 13.51+/-5.12, and 188.24+/-374.04 for normal healthy adult (beta(A)/beta(A),alphaalpha/alphaalpha), infant (beta(A)/beta(A),alphaalpha/alphaalpha), beta- thalassemia minor (beta(T)/beta(A),alphaalpha/alphaalpha) and beta-thalassemia major(beta(T)/beta(T),alphaalpha/alphaalpha) respectively. The alpha/(beta+ gamma ) ratios were 4.43+/-1.17, 0.56+/-0.49, 9.62+/-4.37, and 2.14+/-1.58 for normal healthy adult (beta(A)/beta(A),alphaalpha/alphaalpha), infant (beta(A)/beta(A),alphaalpha/alphaalpha), beta- thalassemia minor (beta(T)/beta(A),alphaalpha/alphaalpha) and beta- thalassemia major(beta(T)/beta(T),alphaalpha/alphaalpha) respectively. The gamma /(beta+ gamma ) ratios were 0.04+/-0.03, 0.92+/-0.06, 0.28+/-0.18, and 0.95+/-0.04 for normal healthy adult (beta(A)/beta(A),alphaalpha/alphaalpha), infant (beta(A)/beta(A),alphaalpha/alphaalpha), beta- thalassemia minor (beta(T)/beta(A),alphaalpha/alphaalpha) and beta- thalassemia major (beta(T)/beta(T),alphaalpha/alphaalpha) respectively. Following statistical analyses, the alpha/beta and alpha/(beta+ gamma ) globin gene mRNA ratios were significantly different among four different groups (normal adult, normal infant, beta- thalassemia minor and beta- thalassemia major). The gamma /(beta + gamma ) globin gene mRNA ratio was significantly different among all groups except for between infant and beta- thalassemia major patients. Human beta globin gene mRNA levels decrease progressively and dramatically from normal adults to beta-thalassemia patients with beta-thalassemia major having the lowest levels. On the other hand, the gamma globin gene mRNA levels increase progressively from normal adult to beta-thalassemia patients with beta-thalassemia major having the highest levels. Infants have relatively lower levels of beta but higher levels of gamma globin gene mRNA as compared to those in normal adults. Thus, the relative expression levels of alpha, beta or gamma globin genes varied but inter-related among different ages of normal individuals and different beta-thalassemia genotypes.