改良2-^(△△CT)法分析RNASET2基因在卵巢癌卡铂耐药中的表达

Analysis of RNASET2 Expression in Carboplatin-resistant Human Ovarian Carcinoma Cells Using A Modified 2^(-△△CT) Method in Real-time PCR

目的:建立改良的相对定量2^(-△△CT)法并应用其分析RNASET2基因在卵巢癌卡铂耐药细胞系中的差异表达。方法:分别提取卵巢癌卡铂耐药SKOV3细胞系与其亲本SKOV3细胞系的总RNA,将等量的mRNA逆转录为cDNA,应用实时荧光定量PCR(real-time RT-PCR)技术检测核糖核酸酶T2(RNASET2)基因在卵巢癌卡铂耐药中的差异表达,参照2^(-△△CT)公式的推导步骤,对其改良演化出一个新的公式即改良2^(-△△CT)公式。以绝对定量法为对照,应用该公式分析real-time RT-PCR技术检测结果并比较两种方法的差异。结果:改良2^(-△△CT)公式与绝对定量法分析实验结果差异无统计学意义(P>0.05)。卵巢癌卡铂耐药SKOV3细胞系RNASET2基因的表达相对于卵巢癌非耐药SKOV3细胞系的表达降低。结论:改良2^(-△△CT)公式分析real-time RT-PCR反应数据是一种简便可行的相对定量方法,RNASET2表达下调与卵巢癌卡铂耐药相关。

Objective： To develop a modification of the 2^-△△CT method for relative quantification of gene expression and the differential expression of RNASET2 mRNA in earboplatin-resistant SKOV3 human ovarian eareinoma cell line. Methods： The total RNA were extracted from carboplatin-resistant SKOV3 cells and its parental cells, and cDNAs were prepared by reverse transcription （RT） with equal quantity mRNA derivied from these two kinds of cell respectively, and then the differential expressions of RNASET2 were determined by real-time PCR. A new 2^-△△CT formula for relative quantification in real-time PCR, called as a modified 2^-△△CT method,was derived from the deductive procedure of 2^-△△CT formula, and PCR data analysis of differential expressions of RNASET2 mRNA was carried out using this modified 2^-△△CT method, comparing with the absolute quantitative method with double standard curves. Results： Our findings showed that no significant difference was found in PCR data analysis with the two methods （P 〉 0.05） , and that the decreased expression of RNASET2 gene was found in carboplatin-resistant SKOV3 cells but not in SKOV3 eells. Conclusion： This modified 2^-△△CT method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments, and also provides a manageable method for examining large quantities of samples ; A further study will be needed to determine whether the downregtdation of RNASET2 expression may be related to drug resistance of human ovarian carcinoma.