title: Coexpressing shRNA with fluorescence tags for quantification of cell migration studies.
abstract: Understanding migration of cells has many implications in human physiology; some examples include developmental biology, healing, immune responses and tissue remodeling. On the other hand, invasive migration by tumor cells is pathological and is a major cause of mortality amongst cancer sufferers. Cell migration assays have been widely used to quantify potentially metastatic genes. In recent years, the use of RNAi has significantly increased the tools available in cell migration research due to its specific gene targeting for knockdown. The inability to ensure 100% transfection/transduction efficiency reduces the sensitivity of cell migration assays because cells not successfully transfected/transduced with the RNAi are also included in the calculations. This study introduces a different experimental setup mathematically expressed in our named normalized relative infected cell count (N-RICC) that analyses cell migration assays by co-expressing retrovirally transduced shRNA with fluorescence tags from a single vector. Vectors transduced into cells are visible under fluorescence, thus alleviating the problems involved with transduction efficiency by individually identifying cells with targeted genes. Designed shRNAs were targeted against a list of potentially metastatic genes in a highly migratory breast cancer cell line model, MDA-MB-231. We have successfully applied N-RICC analysis to show greater sensitivity of integrin alpha5 (ITGA5) and Ras homologue A (RhoA) in cell metastasis over conventional methods in scratch-wound assays and migration chambers assays.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore 637551, Republic of Singapore.
authors.forename: Christine Xing'er
authors.initials: CX
authors.lastname: Koo
authors.forename: Wanru
authors.initials: W
authors.lastname: Fang
authors.forename: Manuel
authors.initials: M
authors.lastname: Salto-Tellez
authors.forename: David Tai
authors.initials: DT
authors.lastname: Leong
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D054443:Cell Migration Assays; D002465:Cell Movement / Q000235:genetics*; D049109:Cell Proliferation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D039482:Integrin alpha5 / Q000235:genetics*; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D009361:Neoplasm Invasiveness / Q000235:genetics*; D009362:Neoplasm Metastasis / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D020742:rhoA GTP-Binding Protein / Q000235:genetics*; D000097573:Red Fluorescent Protein
chemical_list: D039482:Integrin alpha5; D008164:Luminescent Proteins; D034741:RNA, Small Interfering; D020742:rhoA GTP-Binding Protein; D000097573:Red Fluorescent Protein; C066737:RHOA protein, human