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...


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.



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.


  1. 1.
    St. John TP, Davis RW (1979) Isolation of galactose-inducible DNA sequences fromSachromyces cerevisiaeMby differential plaque filter hybridisation. Cell 16:443.CrossRefPubMedGoogle Scholar
  2. 2.
    Zimmerman CR, Orr WC, Leclerc RF, Barnard EC, Timberlake WE (1980) Molecular cloning and selection of genes regulated inAspergillusdevelopment. Cell 11:709.CrossRefGoogle Scholar
  3. 3.
    Timberlake WE (1980) Developmental gene regulation inAspergillus nidulans. Dev Biol 78:497.CrossRefPubMedGoogle Scholar
  4. 4.
    Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of α-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anaemia. Science 239:1350.CrossRefGoogle Scholar
  5. 5.
    Rappolee DA, Wang A, Mark D, Werb Z (1989) Novel method for studying mRNA phenotypes in single or small numbers of cells. J. Cell Biochem. 39:1–11.CrossRefPubMedGoogle Scholar
  6. 6.
    Brady G, Billia F, Knox J, Hoang T, Kirsch IR, Voura EB, Hawley RG, Cumming R, Buchwald M, Siminovitch K, Miyamoto N, Boehmelt G, Iscove N (1995) Analysis of gene expression in a complex differentiation hierarchy by global amplification of cDNA from single cells. Curr Biol 5:909–922.CrossRefGoogle Scholar
  7. 7.
    Brady G, Barbara M, Iscove NN (1990) Representative in vitro cDNA amplification from individual hemopoietic cells and colonies. Meth Mol Cell Biol 2:17–25.Google Scholar
  8. 8.
    Brady G, Iscove NN (1993) Amplified representative cDNA libraries from single cells. In: Wassarman PM, DePamphilis ML (eds.) Methods in enzymology, vol. 225. Academic, San Diego, pp. 611–623.Google Scholar
  9. 9.
    Trumper LH, Brady G, Bagg A, Gray D, Loke SL, Griesser H, Wagman, R, Braziel R, Gascoyne RD, Vicini, S. (1993) Single-cell analysis of Hodgkin and Reed-Sternberg cells: molecular heterogeneity of gene expression and p53 mutations. Blood 81:3097–3115.PubMedGoogle Scholar
  10. 10.
    Dulac C, Axel R (1995) A novel family of genes encoding putative pheromone receptors in mammals. Cell 83:195–206.CrossRefPubMedGoogle Scholar
  11. 11.
    Cumano A, Paige CJ, Iscove NN, Brady G (1992) Bipotential precursors of B cells and macrophages in murine fetal liver. Nature 356:612–615.CrossRefPubMedGoogle Scholar
  12. 12.
    Cano-Gauci DF, Lualdi JC, Ouellette AJ, Brady G, Iscove NN, Buick RN (1993) In vitro cDNA amplification from individual intestinal crypts: a novel approach to the study of differential gene expression along the cryptvillus axis. Exp Cell Res 208:344–349.CrossRefPubMedGoogle Scholar
  13. 13.
    Barr FG, Emmanuel BS (1990) Application of a subtraction hybridization technique involving photoactivatable biotin and organic extraction to solution hybridization analysis of genomic DNA. Anal Biochem.186:369–373.CrossRefPubMedGoogle Scholar
  14. 14.
    Sugai M, Kondo S, Shimizu A, Honjo T (1998) Isolation of differentially expressed genes upon immunoglobulin class switching by a subtractive hybridization method using uracil DNA glycosylase. Nucleic Acids Res 26:911–918.CrossRefPubMedGoogle Scholar
  15. 15.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar

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

Personalised recommendations