Her2/neu基因定量检测方法的建立

Analysis of Her2/neu Gene by Quantitative RT-PCR

目的建立人Her2／neu基因mRNA荧光定量检测方法，评价该方法的准确性及稳定性。方法基于TaqMan荧光探针技术，构建克隆载体pGEM—T—Her2／neu作为定量模板，通过荧光强度达到一定阈值时的循环数来定量起始模板，以建立实时荧光定量RT—PCR方法；并且在GeneAmp 5700型检测仪上测定10例经免疫组织化学法证实Her2／neu表达卅的乳腺癌组织标本，同时对最大值及最小值重复测量。结果Her2／neu动态检测范围为10^3～10^7拷贝／ugRNA（r≥0．996），内参β-actin的动态检测范围为10^3～10^8拷贝／ugRNA（r〉0.998）。10例检测癌组织的Her2／neu表达范围为6．6×10^4～4．7×10^6拷贝／ug，最大值10次重复测量值为（4．65±0．55）×10^6拷贝／μg，最小值10次重复测量值为（7.36±0.75）×10^4拷贝／μg。结论成功建立人Her2／neu基因表达RT—PCR检测方法，具有较高的准确性和稳定性，可用于检测Her2／neu基因表达水平，为进一步应用于乳腺癌预后判断及术后治疗方案的选择奠定了基础。

Objective To establish a specific fluorogenic quantitative method for detecting the expression of Her2/neu gene in breast cancer and evaluate its stability and correctness. Methods A real-time quantitative reverse transcription polymerase chain reaction（RT-PCR）was set up,based on fluorescent TaqMan methodology. In this method,a prokaryotic expressing vector pGEM-T-Her2/neu was constructed as a standard plasmid. RNA quantification was based on the threshold cycle （Ct）values when using GeneAmp 5700 Sequence Detection Systems to examine the specific expression of Her2/neu in 10 breast cancer specimen confirmed Her2/neu ＋＋＋ by immunohistochemical method. Results The dynamic range of the assay was 10^3- 10^7 copy/big mRNA and that of endogenous standards was 10^8- 10^8copy/big. In 10 breast cancer specimen, the Her2/neu expression ranges from 6.6× 10^4-4.7×10^6 copy/big and the repeated measured values of the maximum and minimum value were（4. 65±0. 55） × 10^6copy/μg and （7. 36±0. 75） × 104 copy/μg, respectively. Conclusion Analysis of Her2/neu gene by quantitative RT-PCR is accurate and stabile which is the first step for the method to be used in selecting the therapeutic regimen and judging the prognosis of breast cancer.