title: Single cell transcriptomic analysis of prostate cancer cells.
abstract: BACKGROUND
  The ability to interrogate circulating tumor cells (CTC) and disseminated tumor cells (DTC) is restricted by the small number detected and isolated (typically <10). To determine if a commercially available technology could provide a transcriptomic profile of a single prostate cancer (PCa) cell, we clonally selected and cultured a single passage of cell cycle synchronized C4-2B PCa cells. Ten sets of single, 5-, or 10-cells were isolated using a micromanipulator under direct visualization with an inverted microscope. Additionally, two groups of 10 individual DTC, each isolated from bone marrow of 2 patients with metastatic PCa were obtained. RNA was amplified using the WT-Ovation™ One-Direct Amplification System. The amplified material was hybridized on a 44K Whole Human Gene Expression Microarray. A high stringency threshold, a mean Alexa Fluor® 3 signal intensity above 300, was used for gene detection. Relative expression levels were validated for select genes using real-time PCR (RT-qPCR).
  
  
  RESULTS
  Using this approach, 22,410, 20,423, and 17,009 probes were positive on the arrays from 10-cell pools, 5-cell pools, and single-cells, respectively. The sensitivity and specificity of gene detection on the single-cell analyses were 0.739 and 0.972 respectively when compared to 10-cell pools, and 0.814 and 0.979 respectively when compared to 5-cell pools, demonstrating a low false positive rate. Among 10,000 randomly selected pairs of genes, the Pearson correlation coefficient was 0.875 between the single-cell and 5-cell pools and 0.783 between the single-cell and 10-cell pools. As expected, abundant transcripts in the 5- and 10-cell samples were detected by RT-qPCR in the single-cell isolates, while lower abundance messages were not. Using the same stringency, 16,039 probes were positive on the patient single-cell arrays. Cluster analysis showed that all 10 DTC grouped together within each patient.
  
  
  CONCLUSIONS
  A transcriptomic profile can be reliably obtained from a single cell using commercially available technology. As expected, fewer amplified genes are detected from a single-cell sample than from pooled-cell samples, however this method can be used to reliably obtain a transcriptomic profile from DTC isolated from the bone marrow of patients with PCa.
authors: Department of Urology, University of Washington, Seattle, WA, USA.
authors: Christopher J
authors: CJ
authors: Welty
authors: Ilsa
authors: I
authors: Coleman
authors: Roger
authors: R
authors: Coleman
authors: Bryce
authors: B
authors: Lakely
authors: Jing
authors: J
authors: Xia
authors: Shu
authors: S
authors: Chen
authors: Roman
authors: R
authors: Gulati
authors: Sandy R
authors: SR
authors: Larson
authors: Paul H
authors: PH
authors: Lange
authors: Bruce
authors: B
authors: Montgomery
authors: Peter S
authors: PS
authors: Nelson
authors: Robert L
authors: RL
authors: Vessella
authors: Colm
authors: C
authors: Morrissey
mesh_terms: D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D009360:Neoplastic Cells, Circulating; D011471:Prostatic Neoplasms / Q000235:genetics*; D012680:Sensitivity and Specificity; D059010:Single-Cell Analysis / Q000379:methods*; D059467:Transcriptome