利用外源RNA作为内参结合qPCR准确检测SF2蛋白RIP富集RNA的效率

Accurate Detection Efficiency of SF2 Protein RIP Enriching RNA Using Exogenous RNA as Reference Gene with qPCR

利用实时荧光定量PCR技术(Real-time Quantitative polymerase chain reaction,q PCR)检测0.1%甲醛交联RNA免疫共沉淀(RNA Immunoprecipitation,RIP)富集SF2蛋白相互作用的RNA效率,为研究可变剪接因子SF2相互作用RNA的富集效率提供准确的质检方案。采用RIP技术获取He La细胞中SF2蛋白相互作用的RNA,等比例加入外源酿酒酵母(BY4741)RNA,并以其β-actin作为内参基因,利用q PCR检测RIP富集SF2蛋白相互作用RNA的效率。结果显示,0.1%甲醛交联RIP技术特异性捕获得到SF2蛋白-RNA复合物;q PCR技术检测阳性基因PABP、Srsf1在SF2抗体捕获的产物和Ig G抗体的产物中相应RNA的浓度差异均在60倍以上。利用q PCR技术,以外源酿酒酵母(BY4741)RNA作为内参,能快速、准确定量检测RNA的富集效率。

Using real-time quantitative polymerase chain reaction （ qPCR ） to detect the efficiency of SF2 enriching RNA based on 0.1% formaldehyde cross-linking RNA immunoprecipitation （ RIP ）, we aim to provide an accurate detection method for studying the enriching efficiency of flexible splicing factor SF2 interacting with RNA. The RNA interacting with SF2 in HeLa cells was acquired with RIP, then RNA of exogenous yeast （ BY4？4 ） in the same ratio was added, and the efficiency of RIP enriching RNA interacting with SF2 was measured via qPCR while using J3-actin as reference gene. Results showed the RNA-SF2 protein complex was successfully obtained based on 0.1% formaldehyde cross-linking immunoprecipitation. The differences of RNA by positive genes （ PABP, Srsfl ） achieved 60 folds in SF2 and IgG samples via qPCR. Conclusively, qPCR combined with exogenous yeast （ BY474 ） as reference gene may expeditiously and accurately quantify the RNA-enriched efficiency for SF2.