title: Streamlining gene expression analysis: integration of co-culture and mRNA purification.
abstract: Co-culture of multiple cell types within a single device enables the study of paracrine signaling events. However, extracting gene expression endpoints from co-culture experiments is laborious, due in part to pre-PCR processing of the sample (i.e., post-culture cell sorting and nucleic acid purification). Also, a significant loss of nucleic acid may occur during these steps, especially with microfluidic cell culture where lysate volumes are small and difficult to access. Here, we describe an integrated platform for performing microfluidic cell culture and extraction of mRNA for gene expression analysis. This platform was able to recover 30-fold more mRNA than a similar, non-integrated system. Additionally, using a breast cancer/bone marrow stroma co-culture, we recapitulated stromal-dependent, estrogen-independent growth of the breast cancer cells, coincident with transcriptional changes. We anticipate that this platform will be used for streamlined analysis of paracrine signaling events as well as for screening potential drugs and/or patient samples.
authors.affiliation: Departments of Biomedical Engineering, University of Wisconsin-Madison, 1111 Highland Ave., Madison, WI 53705, USA. Berry3@wisc.edu.
authors.forename: Scott M
authors.initials: SM
authors.lastname: Berry
authors.forename: Chandresh
authors.initials: C
authors.lastname: Singh
authors.forename: Jessica D
authors.initials: JD
authors.lastname: Lang
authors.forename: Lindsay N
authors.initials: LN
authors.lastname: Strotman
authors.forename: Elaine T
authors.initials: ET
authors.lastname: Alarid
authors.forename: David J
authors.initials: DJ
authors.lastname: Beebe
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018920:Coculture Techniques / Q000295:instrumentation / Q000379:methods*; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D019899:Paracrine Communication / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000166:cytology*
chemical_list: D012333:RNA, Messenger