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《Hereditas(Beijing)》 2005-01
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Quantitative Analysis of Human Globin Gene Expression in β-thalassemia Using Real-Time RT-PCR

HAN Jun-Ying~1, ZENG Rui-Ping~1, CHENG Gang~2, HU Bin~1, LI Hu~2, LAI Yong-Rong~3(1. Department of Medical Genetics, Sun Yat-sen Medical College, Sun Yat-Sen University, Guangzhou 510080, China;2. Da-An Gene Diagnostic Center, Sun Yat-Sen University, Guangzhou 510080, China;3. Affiliated First Hospital, Guangxi University of Medical Sciences, Nanning 530000, China)  
Whole blood samples were collected from 100 normal healthy adults, from umbilical cord of 33 newborn infants, 111 individuals with β-thalassemia minor (β~T/β~A,αα/αα) and 39 with β-thalassemia major (β~T/β~T,αα/αα). Prior to quantitative analysis of globin gene expression, DNA was extracted from all blood samples and used for β-thalassemia genotype analysis. Different types of β 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 α, β and γ globin mRNA using three sets of primers and fluorescent-labeled probes, designed according to the sequences of α, β and γ human globin gene. Real-time RT-PCR was performed in ABI 7700 system. Following the real-time RT-PCR, the mean values of α, β and γ globin mRNA were calculated and the ratios of α/β, α/(β+γ) and γ/(β+γ) were determined to characterize the relative expression levels of different globin genes among normal adult, infant, β-thalassemia minor and β-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 β-thalassemia patients. Due to vast variations of the mean globin gene mRNA levels among different groups, log conversion of α/β+1, α/(β+γ)+1 and γ/(β+γ) +1 was used for statistical analyses and intergroup comparison. The α/β 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 (β~A/β~A,αα/αα), infant (β~A/β~A,αα/αα), β-thalassemia minor (β~T/β~A,αα/αα) and β-thalassemia major(β~T/β~T,αα/αα) respectively. The α/(β+γ) ratios were 4.43±1.17, 0.56±0.49, 9.62±4.37, and 2.14±1.58 for normal healthy adult (β~A/β~A,αα/αα), infant (β~A/β~A,αα/αα), β-thalassemia minor (β~T/β~A,αα/αα) and β-thalassemia major(β~T/β~T,αα/αα) respectively. Theγ/(β+γ) ratios were 0.04±0.03, 0.92±0.06, 0.28±0.18, and 0.95±0.04 for normal healthy adult (β~A/β~A,αα/αα), infant (β~A/β~A,αα/αα), β-thalassemia minor (β~T/β~A,αα/αα) and β-thalassemia major(β~T/β~T,αα/αα) respectively. Following statistical analyses, the α/β and α/(β+γ) globin gene mRNA ratios were significantly different among four different groups (normal adult, normal infant, β-thalassemia minor and β-thalassemia major). The γ/(β+γ) globin gene mRNA ratio was significantly different among all groups except for between infant and β-thalassemia major patients. Human β globin gene mRNA levels decrease progressively and dramatically from normal adults to β-thalassemia patients with β-thalassemia major having the lowest levels. On the other hand, the γ globin gene mRNA levels increase progressively from normal adult to β-thalassemia patients with β-thalassemia major having the highest levels. Infants have relatively lower levels of β but higher levels of γ globin gene mRNA as compared to those in normal adults. Thus, the relative expression levels of α, β or γ globin genes varied but inter-related among different ages of normal individuals and different β-thalassemia genotypes.
【Fund】: 广东省卫生厅"五个一"重点科研基金资助项目(522202009);; "211工程"资助项目(201038)~~
【CateGory Index】: R446.9
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