Matching Items (4)

137491-Thumbnail Image.png

A Practical Method for Single Cell Isolation and Analysis by RT-qPCR and Fluorescence Microscopy

Description

Single cell heterogeneity plays an important role in the onset and progression of a variety of disease pathologies. One of the most notable examples of the impact of heterogeneity in the complexity of a disease is cancer. Traditionally, molecular

Single cell heterogeneity plays an important role in the onset and progression of a variety of disease pathologies. One of the most notable examples of the impact of heterogeneity in the complexity of a disease is cancer. Traditionally, molecular analyses on cancer-related samples have been performed on bulk populations of cells, with the resultant data only representative of an average of the population, thereby concealing potentially relevant information about individual cells. Performing these studies at the single cell level is proposed to address this issue. However, current methods for the isolation and analysis of single cells often require specialized and expensive equipment that may be prohibitive to labs wishing to perform such analyses. Herein, a method for the isolation and gene expression analysis of single cells is described that (1) relies only on readily available, inexpensive materials, (2) is compatible with phase and fluorescent microscopy, and (3) allows for the ability to track specific cells throughout all measurements. This method utilizes random seeding of single cells on 72-well Terasaki plates (also called microtest plates) that have 20 µl, optically clear flat-bottomed wells in order to circumvent the need for specific hardware for cell isolation. Suspensions of the Barrett’s esophagus epithelial cell line CP-D stably expressing turboGFP and a related, GFP-negative BE cell line, CP-A, were prepared, seeded at a concentration of approximately 1-2 cells/well and incubated overnight. Wells containing single cells were visually identified using phase-contrast and fluorescent microscopy. Single cells were then lysed directly in the well, total RNA was isolated, and RT-qPCR was performed. RT-qPCR results reflected the ability to distinguish between turboGFP-expressing and non-expressing cells that matched previous identification by microscopy. These results indicate that this is a convenient and cost-effective method for studying gene expression in single cells.

Contributors

Agent

Created

Date Created
2013-05

137396-Thumbnail Image.png

Single-Cell Gene Expression in Esophageal Adenocarcinoma

Description

Esophageal adenocarcinoma (EAC) is one of the most lethal and fastest growing cancers in the United States. Its onset is commonly triggered by metaplastic transformation of normal squamous esophageal epithelial cells to Barrett's esophagus (BE) cells in response to acid

Esophageal adenocarcinoma (EAC) is one of the most lethal and fastest growing cancers in the United States. Its onset is commonly triggered by metaplastic transformation of normal squamous esophageal epithelial cells to Barrett's esophagus (BE) cells in response to acid reflux. BE patients are believed to progress through non-dysplastic metaplasia and increasing grades of dysplasia prior to EAC. Conventional cancer diagnostic tools rely on bulk-cell analyses that are incapable of identifying intratumoral heterogeneity or rare driver cells that play important roles in cancer progression. An improved single-cell method of cancer diagnosis would overcome this challenge by detecting cancer initiating cells before they progress into untreatable stages. In this study, using EAC as a model, we attempted to identify a more effective method of cancer diagnosis. We quantified the single- and bulk-cell mRNA expression of genes that have been proposed to be instrumental in the progression of EAC through BE. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) analysis was performed on human primary cells to measure the mRNA expression levels of BE- and EAC-associated genes. Our results showed high levels of heterogeneity of CDX2 and TFF3 at the single-cell resolution in human BE and EAC samples. Additionally, while expression of VEGF is generally low at the bulk-cell level, our results showed that a few, rare cells had significantly higher VEGF expression levels than the majority of cells in the EAC sample. In conclusion, we have affirmed that EAC cancer cells, as well as BE cells, show high levels of heterogeneity. Based on the VEGF gene expression pattern, single-cell analysis could potentially be more effective for identifying rare, but essential cells for cancer progression, which could then be targeted for treatment. Future studies will focus on analyzing human samples from thousands of normal and cancer subjects to validate the use of single-cell profiling in cancer.

Contributors

Agent

Created

Date Created
2013-12

152402-Thumbnail Image.png

Insulator based dielectrophoretic trapping of single mammalian cells

Description

This work demonstrated a novel microfluidic device based on direct current (DC) insulator based dielectrophoresis (iDEP) for trapping individual mammalian cells in a microfluidic device. The novel device is also applicable for selective trapping of weakly metastatic mammalian breast cancer

This work demonstrated a novel microfluidic device based on direct current (DC) insulator based dielectrophoresis (iDEP) for trapping individual mammalian cells in a microfluidic device. The novel device is also applicable for selective trapping of weakly metastatic mammalian breast cancer cells (MCF-7) from mixtures with mammalian Peripheral Blood Mononuclear Cells (PBMC) and highly metastatic mammalian breast cancer cells, MDA-MB-231. The advantage of this approach is the ease of integration of iDEP structures in microfliudic channels using soft lithography, the use of DC electric fields, the addressability of the single cell traps for downstream analysis and the straightforward multiplexing for single cell trapping. These microfluidic devices are targeted for capturing of single cells based on their DEP behavior. The numerical simulations point out the trapping regions in which single cell DEP trapping occurs. This work also demonstrates the cell conductivity values of different cell types, calculated using the single-shell model. Low conductivity buffers are used for trapping experiments. These low conductivity buffers help reduce the Joule heating. Viability of the cells in the buffer system was studied in detail with a population size of approximately 100 cells for each study. The work also demonstrates the development of the parallelized single cell trap device with optimized traps. This device is also capable of being coupled detection of target protein using MALDI-MS.

Contributors

Agent

Created

Date Created
2013

157181-Thumbnail Image.png

Highly Multiplexed Single Cell in situ Protein Analysis with Cleavable Fluorescent Probes

Description

Measurements of different molecular species from single cells have the potential to reveal cell-to-cell variations, which are precluded by population-based measurements. An increasing percentage of researches have been focused on proteins, for its central roles in biological processes. Immunofluorescence (IF)

Measurements of different molecular species from single cells have the potential to reveal cell-to-cell variations, which are precluded by population-based measurements. An increasing percentage of researches have been focused on proteins, for its central roles in biological processes. Immunofluorescence (IF) has been a well-established protein analysis platform. To gain comprehensive insights into cell biology and diagnostic pathology, a crucial direction would be to increase the multiplexity of current single cell protein analysis technologies.

An azide-based chemical cleavable linker has been introduced to design and synthesis novel fluorescent probes. These probes allow cyclic immunofluorescence staining which leads to the feasibility of highly multiplexed single cell in situ protein profiling. These highly multiplexed imaging-based platforms have the potential to quantify more than 100 protein targets in cultured cells and more than 50 protein targets in single cells in tissues.

This approach has been successfully applied in formalin-fixed paraffin-embedded (FFPE) brain tissues. Multiplexed protein expression level results reveal neuronal heterogeneity in the human hippocampus.

Contributors

Agent

Created

Date Created
2019