【原】肺癌内参基因如何选择？组队内参来一把？

小小医生孙丹雄 2023-03-28 发布于云南

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常记溪亭日暮，沉醉不知归路。

公众号：小小医生之有趣的医学

前言

无言！

肺癌研究如何选择内参，大家都知道，βactin、GAPDH。肺癌研究如何选择最佳内参，大家都不知道。

白晓燕,林嘉颖,吴一龙（大咖）.肺组织标本荧光定量PCR内参基因精确性鉴定[J].肿瘤研究与临床,2010。

Identification of the reference genes' accuracy for lung tissue specimens by real-time quantitative PCR

摘要：

目的

分析比较广泛应用的内参基因β-葡萄糖苷酸酶(GUSB)、3-磷酸甘油醛脱氢酶(GAPDH)和β2-微球蛋白(β 2-M)在中国人的肺癌组织及正常肺组织中的表达稳定性.

方法

采用实时定量PCR技术(qRT-PCR)检测这3个看家基因在20例肺癌组织及相应癌旁正常肺组织中的表达情况,并用两款国际通用的内参基因筛选软件GeNorm和NormFinder分析其稳定性.

结果

GAPDH在组内及组间的表达差异均最大[肿瘤与正常组内标准差(s)分别为1.07和0.93;|△Ct|=2.01±1.06;P=0.000].用两个软件分析均以3个基因的平均值做内参最为稳定,但t检验结果显示以GUSB和β2-M二者平均值作为内参的组内及组间表达差异最小,并且是唯一一个组合在肿瘤和正常组间差异无统计学意义(肿瘤与正常组内s分别为0.53和0.79;|△Ct|=0.73±0.53;P=0.053).

结论

3个看家基因均不适于单独作为肺组织荧光定量PCR检测的内参;GUSB和β2-M的均值是这3个基因不同组合中作为肺组织标本内参的最好选择.

abstracts：

Objective To analyze the expression stability of the three widely used reference genes β-glueuronidase (GUSB), glycera]dehydes-3-phosphate dehydrogenase (GAPDH), β2-microglobulin (β2-M) in Chinese lung cancer tissue specimens and normal lung tissue specimens.

Methods Gene expression wasmeasured by quantitative real time PCR and expression stability was analyzed with two widely used softwares genorm and normfinder.

Results The intra-and inter-group difference of GAPDH is maximum (The intra and inter-group s is 1.07 and 0.93 respectively, |△ Ct|=2.01±1.06; P =0.000). The mean of these three genes' Ct value is the most stable one analyzed by the two softwares. But the t test showed that the mean of Ct value of GUSB and β2-M is the unique combination that had the minimum intra-and inter-group difference, with no statistically significant differences between normal and malignant samples (The intra-and inter-group s is 0.53 and 0.79 respective]y, |△Ct|= 0.73±0.53; P =0.053).

Conclusion It is inappropriate to normalize data derived from lung tissue specimens using one of these three housekeeping genes alone. Among the different combinations of these three genes, the mean of the Ct values of GUSB and β2-M is the best choice as the internal control of lung tissue specimens.