Abstract
Here, we describe a protocol for fluorescence-activated cell sorting (FACS) of human EpCAM+ cells from fresh surgically resected specimens. We then use Q-PCR to identify specific molecular targets associated with the metastatic phenotype. This combined approach enables a qualitative and quantitative gene expression analysis of lung cancer samples. We describe how to use the protocol for Rac GEFs, but it can be applied broadly to other molecular targets.
For complete details on the use and execution of this protocol, please refer to Cooke et al. (2021) and Quatromoni et al. (2015).
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•Tumoral and adjacent non-tumoral human lung tissue enzymatic digestion protocol•Fluorescence-activated cell sorting of EpCAM+ cells from surgically resected specimens•Q-PCR analysis of specific molecular targets associated with the metastatic phenotype•Rac-GEF expression analysis in isolated human lung cancer EpCaM+ cells
Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Here, we describe a protocol for fluorescence-activated cell sorting (FACS) of human EpCAM+ cells from fresh surgically resected specimens. We then use Q-PCR to identify specific molecular targets associated with the metastatic phenotype. This combined approach enables a qualitative and quantitative gene expression analysis of lung cancer samples. We describe how to use the protocol for Rac GEFs, but it can be applied broadly to other molecular targets.