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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 establish a modified relative quantitative 2 ~ (-ΔΔ CT) method and to analyze the differential expression of RNASET2 gene in carboplatin-resistant cell lines of ovarian cancer. Methods: Total RNA was extracted from ovarian cancer cell line SKOV3 and its parental SKOV3 cell line respectively, and the same amount of mRNA was reverse transcribed into cDNA. Real-time RT-PCR was used to detect the expression of ribonuclease The differential expression of T2 (RNASET2) gene in the resistance to carboplatin in ovarian cancer was studied. According to the derivation procedure of 2 ~ (- △△ CT) formula, a new formula was developed to improve the 2 ~ (- △△ CT) formula. The absolute quantitative method as a control, the application of this formula analysis of real-time RT-PCR test results and compare the differences between the two methods. Results: There was no significant difference between the modified 2 ~ (-ΔΔ CT) formula and the absolute quantitative analysis (P> 0.05). The expression of RNASET2 gene in ovarian cancer carboplatin-resistant SKOV3 cell line was decreased relative to that in ovarian cancer non-resistant SKOV3 cell line. Conclusion: The improved 2 ~ (-ΔΔCT) formula is a simple and feasible method for relative quantitative analysis of real-time RT-PCR data. The down-regulation of RNASET2 is associated with carboplatin resistance in ovarian cancer.