1. Streamlining gene expression analysis: integration of co-culture and mRNA purification
- Author
-
David J. Beebe, Chandresh Singh, Jessica D. Lang, Lindsay N. Strotman, Scott M. Berry, and Elaine T. Alarid
- Subjects
Microfluidics ,Biophysics ,Paracrine Communication ,Breast Neoplasms ,Biology ,Biochemistry ,Article ,Paracrine signalling ,Cell Line, Tumor ,Gene expression ,Humans ,RNA, Messenger ,Cell Proliferation ,Cell growth ,Reverse Transcriptase Polymerase Chain Reaction ,Gene Expression Profiling ,Nucleic acid methods ,Cell sorting ,Molecular biology ,Coculture Techniques ,Cell biology ,Gene expression profiling ,Cell culture ,Female ,Stromal Cells - 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.
- Published
- 2014