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Yeast
Volume 1, Issue 3, Pages 201-210
http://dx.doi.org/10.1002/1097-0061(20000930)17:3<201::AID-YEA30>3.0.CO;2-R
Research Article

Global cDNA Amplification Combined with Real-Time RT–PCR: Accurate Quantification of Multiple Human Potassium Channel Genes at the Single Cell Level

1School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK
2G.38 Stopford Building, School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK

Received 23 June 2000; Accepted 19 July 2000

Copyright © 2000 Hindawi Publishing Corporation. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

We have developed a sensitive quantitative RT–PCR procedure suitable for the analysis of small samples, including single cells, and have used it to measure levels of potassium channel mRNAs in a panel of human tissues and small numbers of cells grown in culture. The method involves an initial global amplification of cDNA derived from all added polyadenylated mRNA followed by quantitative RT–PCR of individual genes using specific primers. In order to facilitate rapid and accurate processing of samples, we have adapted the approach to allow use of TaqMan real-time quantitative PCR. We demonstrate that the approach represents a major improvement over existing conventional and real-time quantitative PCR approaches, since it can be applied to samples equivalent to a single cell, is able to accurately measure expression levels equivalent to less than 1/100th copy/cell (one specific cDNA molecule present amongst 108 total cDNA molecules). Furthermore, since the initial step involves a global amplification of all expressed genes, a permanent cDNA archive is generated from each sample, which can be regenerated indefinitely for further expression analysis.