Cells are often characterized by their gene expression profile. However, commonly used methods to detect mRNA require cell pooling and could therefore mask differences in gene expression within heterogeneous cell populations. q2PISH allows for the analysis of both qualitative and quantitative (q2) gene expression on cultured cells for quality control measures with single cell resolution. q2PISH was optimized for the subsequent use of two alkaline phosphatase substrates in combination with a cell nucleus count to allow for accurate quantification of gene expression per cell and simultaneously qualitative assessment of potential culture population drift or heterogeneity. As proof of principle the assay was applied to cell lines derived from different areas of the bovine intervertebral disc, showing significant difference in the expression of Col1a1, Col2a1, Acan and Sox9. Furthermore, the assay served to explore a potential impact on cultured cells when substituting a critical media component, fetal bovine serum (FBS), suggesting no significant difference in gene expression for the biomarkers analyzed. As a tool, q2PISH serves as an accurate quality control with single cell resolution for cultured cells.
Alkaline phosphatase Bovine NBT/BCIP PISH pNPP RNA in situ hybridization Single cell gene expression
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We are grateful to Peter Braun of Woodcrest Dairy (Lisbon, NY), Willard and Sons (Heuvelton, NY) and Tritown Meat Packing (Brasher Falls, NY) for providing us with bovine tissue for cell line derivation. This work was supported by the Bayard and Virginia Clarkson Endowment Fund to Thomas Lufkin. Rachel Yerden was supported by the Collegiate Science and Technology Entry Program (CSTEP) and Community of Underrepresented Professional Opportunities (CUPO) programs.
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