From phenotypical investigation to RNA-sequencing for gene expression analysis: A workflow for single and pooled rare
Tania Rossi1, Davide Angeli2, Giovanni Martinelli3
1Biosciences Laboratory, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) "Dino Amadori", Meldola, Italy.
Abstract:
Combining phenotypical and molecular characterization of rare cells is challenging due to their scarcity and difficult handling. In oncology, circulating tumor cells (CTCs) are considered among the most important rare cell populations. Their phenotypic and molecular characterization is necessary to define the molecular mechanisms underlying their metastatic potential. Several approaches that require cell fixation make difficult downstream molecular investigations on RNA. Conversely, the DEPArray technology allows phenotypic analysis and handling of both fixed and unfixed cells, enabling a wider range of applications. Here, we describe an experimental workflow that allows the transcriptomic investigation of single and pooled OE33 cells undergone to DEPArray analysis and recovery. In addition, cells were tested at different conditions (unfixed, CellSearch fixative (CSF)- and ethanol (EtOH)-fixed cells). In a forward-looking perspective, this workflow will pave the way for novel strategies to characterize gene expression profiles of rare cells, both single-cell and low-resolution input.
Insights
Investigating rare cells like circulating tumor cells (CTCs) is difficult. A new workflow using DEPArray technology enables transcriptomic analysis of single or pooled CTCs after phenotypic characterization.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Characterizing rare cells, such as circulating tumor cells (CTCs), is crucial for understanding cancer metastasis.
- Existing methods often hinder downstream molecular analysis, particularly RNA studies, due to cell fixation requirements.
Purpose of the Study:
- To develop and validate an experimental workflow for transcriptomic analysis of rare cells.
- To enable phenotypic and molecular characterization of single or pooled cells using DEPArray technology.
Main Methods:
- DEPArray technology was employed for phenotypic analysis and cell recovery.
- OE33 cells were analyzed under various fixation conditions (unfixed, CellSearch fixative, ethanol).
- Transcriptomic analysis was performed on single and pooled recovered cells.
Main Results:
- The described workflow successfully enabled transcriptomic investigation of single and pooled OE33 cells post-DEPArray analysis.
- The method is applicable to cells processed under different fixation conditions, including unfixed, CSF-fixed, and EtOH-fixed cells.
Conclusions:
- This workflow facilitates comprehensive characterization of rare cells, combining phenotypic and molecular data.
- It offers a versatile approach for gene expression profiling of rare cells, supporting future research in cancer biology and diagnostics.
