Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1993 Jul 11;21(14):3159–3166. doi: 10.1093/nar/21.14.3159

Variation in cellular EGF receptor mRNA expression demonstrated by in situ reverse transcriptase polymerase chain reaction.

B W Heniford 1, A Shum-Siu 1, M Leonberger 1, F J Hendler 1
PMCID: PMC309750  PMID: 7688113

Abstract

Cell to cell variation of epidermal growth factor (EGF) receptor mRNA levels in heterogeneous tissues has been demonstrated with an in situ assay that couples reverse transcriptase with the polymerase chain reaction (in situ RT-PCR). EGF receptor mRNA is consistently more highly expressed in regions where cell division occurs; EGF receptor mRNA is markedly reduced if not absent in areas of squamous cell differentiation. Both human and mouse tumors overexpress EGF receptor mRNA when compared to normal tissue. In situ RT-PCR performed on thin sections obtained from cell pellets of cultured cells with known levels of EGF receptor mRNA expression demonstrated that the mRNA detected is consistent with that observed by Northern analysis and quantitative PCR on isolated RNA and by protein levels detected by antibody binding assays. In situ RT-PCR is significantly more sensitive than in situ hybridization (ISH). The method avoids background associated with hybridization reactions as in ISH or ISH following in situ PCR. In situ RT-PCR appears to be applicable to any gene as long as the oligonucleotide primers used have been proven to be specific and effective in a standard RT-PCR assay.

Full text

PDF
3159

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Avivi A., Lax I., Ullrich A., Schlessinger J., Givol D., Morse B. Comparison of EGF receptor sequences as a guide to study the ligand binding site. Oncogene. 1991 Apr;6(4):673–676. [PubMed] [Google Scholar]
  2. Diedrich U., Baron E., Behnke J., Zoll B. Amplification and expression of the epidermal growth factor receptor gene during the progression of neuroepithelial tumours in vivo. J Neurol. 1992 Oct;239(8):465–468. doi: 10.1007/BF00856813. [DOI] [PubMed] [Google Scholar]
  3. Eisbruch A., Blick M., Lee J. S., Sacks P. G., Gutterman J. Analysis of the epidermal growth factor receptor gene in fresh human head and neck tumors. Cancer Res. 1987 Jul 1;47(13):3603–3605. [PubMed] [Google Scholar]
  4. Embleton M. J., Gorochov G., Jones P. T., Winter G. In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells. Nucleic Acids Res. 1992 Aug 11;20(15):3831–3837. doi: 10.1093/nar/20.15.3831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gusterson B., Cowley G., Smith J. A., Ozanne B. Cellular localisation of human epidermal growth factor receptor. Cell Biol Int Rep. 1984 Aug;8(8):649–658. doi: 10.1016/0309-1651(84)90045-6. [DOI] [PubMed] [Google Scholar]
  6. Haase A. T., Retzel E. F., Staskus K. A. Amplification and detection of lentiviral DNA inside cells. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4971–4975. doi: 10.1073/pnas.87.13.4971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hendler F. J., Ozanne B. W. Human squamous cell lung cancers express increased epidermal growth factor receptors. J Clin Invest. 1984 Aug;74(2):647–651. doi: 10.1172/JCI111463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Li H. H., Gyllensten U. B., Cui X. F., Saiki R. K., Erlich H. A., Arnheim N. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature. 1988 Sep 29;335(6189):414–417. doi: 10.1038/335414a0. [DOI] [PubMed] [Google Scholar]
  9. Nuovo G. J., MacConnell P., Forde A., Delvenne P. Detection of human papillomavirus DNA in formalin-fixed tissues by in situ hybridization after amplification by polymerase chain reaction. Am J Pathol. 1991 Oct;139(4):847–854. [PMC free article] [PubMed] [Google Scholar]
  10. Spann W., Pachmann K., Zabnienska H., Pielmeier A., Emmerich B. In situ amplification of single copy gene segments in individual cells by the polymerase chain reaction. Infection. 1991 Jul-Aug;19(4):242–244. doi: 10.1007/BF01644953. [DOI] [PubMed] [Google Scholar]
  11. Ullrich A., Coussens L., Hayflick J. S., Dull T. J., Gray A., Tam A. W., Lee J., Yarden Y., Libermann T. A., Schlessinger J. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. 1984 May 31-Jun 6Nature. 309(5967):418–425. doi: 10.1038/309418a0. [DOI] [PubMed] [Google Scholar]
  12. Wong A. J., Bigner S. H., Bigner D. D., Kinzler K. W., Hamilton S. R., Vogelstein B. Increased expression of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with gene amplification. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6899–6903. doi: 10.1073/pnas.84.19.6899. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES