Abstract
Defining biological signals at the single cell level can identify cancer initiating driver mutations. Techniques to isolate single cells such as microfluidics sorting and magnetic capturing systems have limitations such as: high cost, labor intense, and the requirement of a large number of cells. Therefore, the goal of our current study is to identify a cost and labor effective, reliable, and reproducible technique that allows single cell isolation for analysis to promote regular laboratory use, including standard reverse transcription PCR (RT-PCR).In the current study, we utilized single prostate cells isolated from the CWR-R1 prostate cancer cell line and human prostate clinical specimens, based on the ATP binding cassette (ABC) transporter efflux of dye cycle violet (DCV), side population assay. Expression of four genes: ABCG2; Aldehyde dehydrogenase1A1 (ALDH1A1); androgen receptor (AR); and embryonic stem cell marker, Oct-4, were determined.Results from the current study in the CWR-R1 cell line showed ABCG2 and ALDH1A1 gene expression in 67% of single side population cells and in 17% or 100% of non-side population cells respectively. Studies using single cells isolated from clinical specimens showed that the Oct-4 gene is detected in only 22% of single side population cells and in 78% of single non-side population cells. Whereas, AR gene expression is in 100% single side population and non-side population cells isolated from the same human prostate clinical specimen.These studies show that performing RT-PCR on single cells isolated by FACS can be successfully conducted to determine gene expression in single cells from cell lines and enzymatically digested tissue. While these studies provide a simple yes/no expression readout, the more sensitive quantitative RT-PCR would be able to provide even more information if necessary.
Highlights
Single cell approaches are becoming increasingly useful because intra- and inter-tumoral genomic heterogeneity is difficult to analyze when studied in bulk cell populations [1, 2]
Results from the current study demonstrated that ATP binding cassette transporter G2 (ABCG2), Aldehyde dehydrogenase1A1 (ALDH1A1), and Oct-4 genes are expressed heterogeneously in single cells isolated from the CWR-R1 prostate cancer cell line (Table 3), a difference in gene expression would be hard to detect when analyzing bulk cells
We demonstrated a technique involving a series of steps which enabled the isolation of single cells to identify gene expression in a single side population or a single non-side population cell
Summary
Single cell approaches are becoming increasingly useful because intra- and inter-tumoral genomic heterogeneity is difficult to analyze when studied in bulk cell populations [1, 2]. Analysis of single cells is being conducted in the area of diabetes, where in a study the authors performed a novel real-time PCR assay in single cells isolated from pancreas in order to identify the mechanisms leading to progression of type 1 diabetes [14]. Analysis of single cells from human sperm cells and embryos is performed to identify presence of genetic diseases [11, 15] Sophisticated techniques such as microarray expression assay, RNA sequencing, whole transcriptomic analysis (WTA) and whole genomic analysis (WGA) of single cells are well developed and are being used to study the genomic and transcriptomic variability in single cells from different cell types [7, 16, 17]. The ability to measure gene expression in single cells can be helpful in understanding the mechanisms leading to disease progression and to develop better therapeutic regimen in cancer and for many other diseases
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