两种荧光定量聚合酶链反应法检测乳腺癌组织中微小RNA-21的应用分析

Comparison of microRNA-21 Detection Results in Breast Cancer Tissues with Two Fluorescent Quantitative PCR Methods

目的比较两种荧光定量聚合酶链反应(FQ-PCR)法检测乳腺癌组织中微小RNA(miR)-21的表达,选择更适用于临床的检测方法。方法选取遵义医学院附属医院普外科2010年度住院的手术治疗的乳腺癌患者,手术切除的乳腺组织标本,包括乳腺癌组织和癌旁组织(距肿瘤边缘>5 cm)14对。分别用SYBR GreenⅠ染料和Taq-Man探针作为荧光指示剂,FQ-PCR法检测乳腺癌组织和癌旁组织中miR-21的相对表达水平,应用SPSS 15.0统计软件分析两种检测模式的差异。最后用Northern Blot法进行实验验证。结果 SYBR GreenⅠ染料法检测乳腺癌组织与癌旁组织中miR-21的表达,差异有统计学意义(t=6.704,P=0.000);TaqMan探针法检测乳腺癌组织与癌旁组中miR-21的表达,差异有统计学意义(t=7.238,P=0.000)。SYBR GreenⅠ染料法与TaqMan探针法检测乳腺癌组织中miR-21的表达,差异无统计学意义(t=2.33,P=0.053);SYBR GreenⅠ染料法与TaqMan探针法检测癌旁组织中miR-21的表达,差异无统计学意义(t=0.07,P=0.95)。Northern Blot法检测乳腺癌组织和癌旁组织中miR-21的表达,探针分别为miR-21和U6的反义链,由32P标记。用Image Quant软件测定灰度值评定信号表达强弱,结果表明,乳腺癌组织中miR-21高表达,而癌旁组织中miR-21低表达。结论 SYBR GreenⅠ染料法和TaqMan探针法检测乳腺癌组织miR-21表达均较癌旁组织中明显升高,与Northern Blot法检测结果一致,但SYBR GreenⅠ染料法简单、有效、成本较低,更适用于临床miR-21的检测。

Objective To compare the results of microRNA （miR -21 ） expression in breast cancer tissues detected by two FQ - PCR methods in order to choose a better one in clinic. Methods 14 pairs of breast cancer samples, including cancer tissues and surrounding tissues （ 〉 5 cm from the cancer border） in 2010 were selected. SYBR Green Ⅰ fluorescent dye and Taq- Man probe were used as fluorescent indicator, and quantitative PCR method was used to detect the expression of miR - 21 in breast cancer tissues and surrounding tissues. SPSS 15.0 software was used to analyze the difference of the two methods. Finally, Northern Blot method was used to verify the experiment. Results The expression of miR -21 in breast cancer tissues and sur- rounding tissues detected by SYBR GreenⅠ method and TaqMan probe method both showed statistically significant difference （t =6. 704, P =0. 000; t =7. 238, P =0. 000） . The expression of miR -21 in cancer tissues detected by SYBR Green Ⅰ method and TaqMan method showed no statistically significant difference （ t = 2.33, P = 0. 053）, and the expression of miR - 21 in sur- rounding tissues detected by SYBR Green I method and TaqMan method showed no statistically significant difference （ t = 0. 07, P = 0. 95 ） . Northern Blot method was used to detect the expression of miR - 21 in breast cancer tissues and surrounding tissues and the probes was the antisense strand of miR- 21 and U6 32P was used for marking. Image Quant software was used to detect signal by testing grey value and the results showed that miR - 21 had high expression in cancer tissues while had low expression in surrounding tissues. Conclusion SYBR Green fluorescent dye method and TaqMan probe method all showed increased expression of miR- 21 in cancer tissues than in surrounding tissues, which is consistent with the results of Northern Blot method. SYBR Green Ⅰ dye method is simple, effective and low cost, so it is more suitable for clinical detection of miR -21.