应用实时定量RT-PCR技术检测b地中海贫血 珠蛋白基因的表达

为了定量检测 b 地中海贫血(b 地贫)的 a、b 和γ珠蛋白基因表达水平， 提取正常成人对照组、正常胎儿对照组和b 地贫患者组组成的样本 DNA，采用反向点杂交法（RDB）分析b 地贫各种突变类型；提取样本RNA用于进行针对a、b 和γ珠蛋白基因的荧光实时定量RT-PCR（FQ RT-PCR）。根据FQ RT-PCR原理，设计合成分别对应于a、b 和γ珠蛋白基因的3对引物和3条荧光探针，FQ RT-PCR在ABI 7700系统进行。用SPSS 10.0对实验数据进行统计学分析，分别计算正常对照组 (bA/bA,aa/aa)，脐带血组(bA/bA,aa/aa)，轻型b 地贫组(bT/bA,aa/aa)，重型b地贫组(bT/bT,aa/aa)的a、b 和γmRNA比值，其中a/b分别为4.62±1.20、7.81±2.89、13.51±5.12、188.24±374.04；a/(b +γ)分别为4.43±1.17、0.56±0.49、9.62±4.37、2.14±1.58；γ/(b+γ) 分别为0.04±0.03、0.92±0.06、0.28±0.18、0.95±0.04。由于组与组之间均值变异范围较大，将其进行对数转换后再进行方差分析。结果表明： a/b与a/(b+γ)在所有组与组之间均有显著性差异。γ/(b+γ)除了在脐带血组和重型b地贫组之间无显著性差异外，在其他组与组之间均有显著性差异。实验说明，人类b珠蛋白基因的表达水平从正常对照组到重型b地贫组急剧下降且以重型b地贫组为最低；相反γ珠蛋白基因表达却明显升高，以重型b地贫组为最高。与正常成人对照组相比，胎儿期b mRNA水平较低但γmRNA 水平较高。因此，正常个体不同时期和不同类型b 地贫之间a、b与γ珠蛋白基因表达不同而且互相影响。 Abstract：whole blood samples were collected from 100 normal healthy adults, from umbilical cord of 33 newborn infants, 111 individuals with b-thalassemia minor (bT/bA,aa/aa) and 39 with b-thalassemia major (bT/bT,aa/aa). Prior to quantitative analysis of globin gene expression, DNA was extracted from all blood samples and used for b-thalassemia genotype analysis. Different types of b 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 a, b andγglobin mRNA using three sets of primers and fluorescent-labeled probes, designed according to the sequences of a, b andγhuman globin gene. Real-time RT-PCR was performed in ABI 7700 system. Following the real-time RT-PCR, the mean values of a, b andγglobin mRNA were calculated and the ratios of a/b, a/(b + γ) andγ/(b + γ) were determined to characterize the relative expression levels of different globin genes among normal adult, infant, b-thalassemia minor and b-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 b-thalassemia patients. Due to vast variations of the mean globin gene mRNA levels among different groups, log conversion of a/b + 1, a/(b + γ) + 1 andγ/(b + γ) +1 was used for statistical analyses and intergroup comparison. The a/b 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 (bA/bA,aa/aa), infant (bA/bA,aa/aa), b- thalassemia minor (bT/bA,aa/aa) and b-thalassemia major(bT/bT,aa/aa) respectively. The a/(b+γ) ratios were 4.43±1.17, 0.56±0.49, 9.62±4.37, and 2.14±1.58 for normal healthy adult (bA/bA,aa/aa), infant (bA/bA,aa/aa), b- thalassemia minor (bT/bA,aa/aa) and b- thalassemia major(bT/bT,aa/aa) respectively. Theγ/(b+γ) ratios were 0.04±0.03, 0.92±0.06, 0.28±0.18, and 0.95±0.04 for normal healthy adult (bA/bA,aa/aa), infant (bA/bA,aa/aa), b- thalassemia minor (bT/bA,aa/aa) and b- thalassemia major(bT/bT,aa/aa) respectively. Following statistical analyses, the a/b and a/(b+γ) globin gene mRNA ratios were significantly different among four different groups (normal adult, normal infant, b- thalassemia minor and b- thalassemia major). The γ/(b + γ) globin gene mRNA ratio was significantly different among all groups except for between infant and b- thalassemia major patients. Human b globin gene mRNA levels decrease progressively and dramatically from normal adults to b-thalassemia patients with b-thalassemia major having the lowest levels. On the other hand, the γglobin gene mRNA levels increase progressively from normal adult to b-thalassemia patients with b-thalassemia major having the highest levels. Infants have relatively lower levels of b but higher levels of γglobin gene mRNA as compared to those in normal adults. Thus, the relative expression levels of a, b or γglobin genes varied but inter-related among different ages of normal individuals and different b-thalassemia genotypes.

Quantitative Analysis of Human Globin Gene Expression in β-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.