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Single-Cell RT-PCR cDNA Subtraction

  • Ebrahim Sakhinia
  • Damian L. Weaver
  • César Núñez
  • Clare Brunet
  • Victoria Bostock
  • Gerard Brady
Part of the METHODS IN MOLECULAR BIOLOGY™ book series (MIMB, volume 461)

1. Introduction

A major problem in trying to understand complex developmental processes is heterogeneity at both the cellular and molecular levels. At the cellular level, it is often difficult to identify cells that are undergoing developmental changes and establish the stage of differentiation they have reached. At the molecular level, there is then a problem in establishing which the many thousands of expressed genes are playing a role in regulating development. Several approaches for identifying expressed candidate developmental regulatory genes are based on comparing the mRNA expression patterns in cells before and after developmental transition points. Differential screening of cDNA libraries with labeled total cDNA probes from contrasting cell samples (1) provides a simple means of identifying genes that are expressed at high levels in one of the samples. The cDNA subtraction protocols (2,3) increased the sensitivity of this type of approach by removing sequences expressed in...

Keywords

cDNA Subtraction Full Speed Driver cDNA Subtraction Protocol Centrifuge Briefly 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

This work was supported by The Leukaemia Research Fund, The Cancer Research Campaign, and The Mark Richardson Memorial Trust. We thank David Masters for contributing to the latter part of this work.

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Copyright information

© Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Ebrahim Sakhinia
    • 1
  • Damian L. Weaver
    • 1
  • César Núñez
    • 1
  • Clare Brunet
    • 1
  • Victoria Bostock
    • 1
  • Gerard Brady
    • 1
  1. 1.School of Biological SciencesUniversity of ManchesterManchesterUK

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