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Quantitative single-cell gene expression measurements of multiple genes in response to hypoxia treatment. Anal Bioanal Chem 2011 Jul;401(1):3-13

Date

05/27/2011

Pubmed ID

21614642

DOI

10.1007/s00216-011-5084-2

Scopus ID

2-s2.0-79960555292   10 Citations

Abstract

Cell-to-cell heterogeneity in gene transcription plays a central role in a variety of vital cell processes. To quantify gene expression heterogeneity patterns among cells and to determine their biological significance, methods to measure gene expression levels at the single-cell level are highly needed. We report an experimental technique based on the DNA-intercalating fluorescent dye SYBR green for quantitative expression level analysis of up to ten selected genes in single mammalian cells. The method features a two-step procedure consisting of a step to isolate RNA from a single mammalian cell, synthesize cDNA from it, and a qPCR step. We applied the method to cell populations exposed to hypoxia, quantifying expression levels of seven different genes spanning a wide dynamic range of expression in randomly picked single cells. In the experiment, 72 single Barrett's esophageal epithelial (CP-A) cells, 36 grown under normal physiological conditions (controls) and 36 exposed to hypoxia for 30 min, were randomly collected and used for measuring the expression levels of 28S rRNA, PRKAA1, GAPDH, Angptl4, MT3, PTGES, and VEGFA genes. The results demonstrate that the method is sensitive enough to measure alterations in gene expression at the single-cell level, clearly showing heterogeneity within a cell population. We present technical details of the method development and implementation, and experimental results obtained by use of the procedure. We expect the advantages of this technique will facilitate further developments and advances in the field of single-cell gene expression profiling on a nanotechnological scale, and eventually as a tool for future point-of-care medical applications.

Author List

Zeng J, Wang J, Gao W, Mohammadreza A, Kelbauskas L, Zhang W, Johnson RH, Meldrum DR

Author

Roger H. Johnson PhD Associate Professor in the Biophysics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Animals
Cell Hypoxia
Cell Line
DNA, Complementary
Epithelial Cells
Esophagus
Gene Expression Profiling
Gene Expression Regulation
Humans
Organic Chemicals
RNA
Reverse Transcriptase Polymerase Chain Reaction
Sensitivity and Specificity
Single-Cell Analysis