实时荧光定量PCR检测人胰岛素样生长因子及其受体基因的方法学构建

Establishment of the real time PCR methods for detecting genes of insulin-like growth factors and their receptors

目的建立检测人胰岛素样生长因子1（insulin-like growth factor-1,IGF-1）、IGF-1受体（IGF-1R）、胰岛素样生长因子2（IGF-2）及IGF-2受体（IGF-2R）基因的实时荧光定量PCR。方法设计并合成检测上述4种基因的引物及TaqMan探针;提取胎盘组织RNA并逆转录为cDNA,分别将扩增的4种基因片段纯化后与质粒载体连接,克隆构建含目的基因片段的重组质粒,分别作为定量检测上述4种基因的标准品;用建立的实时荧光定量PCR检测上述4种基因,并用测序验证。结果 PCR扩增产物经测序分析证实分别为上述4种基因的特异性片段;定量检测IGF-1、IGF-1R、IGF-2及IGF-2R的线性范围分别为1.00×10^2-1.00×10^8copies/μL、2.00×10^2-2.00×10^8copies/μL、3.00×10^2-3.00×10^8copies/μL及5.00×10^2-5.00×10^8copies/μL;相关系数（r）分别为0.994、0.993、0.995及0.996;扩增效率分别为92.35%、96.06%、94.92%及98.84%;批间变异系数（CV）分别为1.64%-2.83%、1.73%-1.98%、2.47%-4.09%及2.25%-2.52%;批内CV分别为1.36%、1.02%、1.04%及0.96%。结论建立了高度敏感、特异检测人IGF及其受体的实时荧光定量PCR法。

Objective To establish the real time PCR methods for detecting the mRNA levels of insulin-like growth factor-1（ IGF-1）, IGF-1 receptor（ IGF-1R）,IGF-2 and IGF-2 receptor（ IGF-2R）. Methods The primers and TaqMan probes for IGF-1,IGF-1R, IGF-2 and IGF-2R were designed and synthesized. Then,the total RNA from placenta tissues was extracted,reversely transcript to cDNA and amplified by PCR with the specific primers for IGF-1,IGF-1R,IGF-2 and IGF-2R. The obtained PCR products were purified and connected with the plasmid vector to construct the recombinant plasmid,which was used as the standard of the established real time PCR. Next,the IGF-1,IGF-1R,IGF-2 and IGF-2R genes were detected by the established real time PCR and verified by DNA sequencing. Results The obtained PCR products were confirmed as the specific fragments of IGF-1,IGF-1R,IGF-2 and IGF-2R by DNA sequencing,respectively. The linear range,the inter-assay coefficient of variation,the intra-assay coefficient of variation,the correlation coefficient and the amplification efficiency were 1. 00 × 10^2- 1. 00 × 10^8copies / μL,1. 64% - 2. 83%,1. 36%,0. 994 and 92. 35% for IGF-1,2. 00 × 10^2- 2. 00 × 10^8copies / μL,1. 73% - 1. 98%,1. 02%,0. 993 and 96. 06% for IGF-1R,3. 00 × 10^2- 3. 00 × 10^8copies / μL,2. 47% - 4. 09%,1. 04%,0. 995 and 94. 92% for IGF-2,and 5. 00 × 10^2- 5. 00 × 10^8copies / μL, 2. 25% - 2. 52%,0. 96%,0. 996 and 98. 84% for IGF-2R,respectively. Conclusion The real time PCR methods with high sensitivity and specificity for detecting the mRNA levels of IGF-1,IGF-1R,IGF-2 and IGF-2R were established successfully.