Advertisement

Biological Procedures Online

, Volume 3, Issue 1, pp 19–25 | Cite as

Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample

  • Maria Marone
  • Simona Mozzetti
  • Daniela De Ritis
  • Luca Pierelli
  • Giovanni Scambia
Open Access
Article
Received:
Accepted:

Abstract

We describe a semiquantitative RT-PCR protocol optimized in our laboratory to extract RNA from as little as 10,000 cells and to measure the expression levels of several target mRNAs from each sample. This procedure was optimized on the human erythroleukemia cell line TF-1 but was successfully used on primary cells and on different cell lines. We describe the detailed procedure for the analysis of Bcl-2 levels. Aldolase A was used as an internal control to normalize for sample to sample variations in total RNA amounts and for reaction efficiency. As for all quantitative techniques, great care must be taken in all optimization steps: the necessary controls to ensure a rough quantitative (semi-quantitative) analysis are described here, together with an example from a study on the effects of TGF-β1 in TF-1 cells.

Indexing terms

RT-PCR mRNA gene expression technique bcl-2 
Download to read the full article text

References

  1. 1.
    PCR technology: principles and applications for DNA amplification. 1989. H.A. Erlich Ed., IRL Press at Oxford Univ. Press, Oxford, UKGoogle Scholar
  2. 2.
    Carding SR., Lu D, Bottomly KA. 1992. A polymerase chain reaction assay for the detection and quantification of cytokine gene expression in small number of cells. J.Immunol.Methods 151, 277–287.PubMedCrossRefGoogle Scholar
  3. 3.
    Marone M, Pierelli L, Mozzetti S, Masciullo V, Bonanno G, Morosetti R, Rutella S, Battaglia A, Rumi C, Mancuso S, Leone G, Scambia, G. 2000. High Cyclin-dependent kinase inhibitors and p53 levels in Bcl-2 and Bcl-xL-expressing primitive proliferating hematopoietic progenitors. Br. J. Haematol. 654–662.Google Scholar
  4. 4.
    Pierelli L, Marone M, Bonanno G, Battaglia A, Mozzetti S, Rumi C, Mancuso S, Leone G, Scambia, G. 2000. Modulation of bcl-2 and p27 in primitive, proliferating hematopoietic progenitors by autocrine TGF-beta1 is a cell cycle independent effect and influences their hematopoietic potential. Blood 95, 3001–3010.PubMedGoogle Scholar
  5. 5.
    Marone M, Ferrandina G, Macchia G, Mozzetti S, De Pasqua A, Benedetti-Panici P, Mancuso S, Scambia G. 2001. Bcl-2, bax, bcl-xL and bcl-xS expression in neoplastic and normal endometrium. Oncology 58, 161–168.CrossRefGoogle Scholar
  6. 6.
    Marone M, Scambia G, Mozzetti S, Ferrandina G, Iacovella S, De Pasqua A, Benedetti-Panici P, Mancuso S. 1998. bcl-2, bax, bcl-xl and bcl-xs expression in normal and neoplastic ovarian tissues. Clinical Cancer Res. 4, 517–524.Google Scholar
  7. 7.
    Marone M, Scambia G, Giannitelli C, Ferrandina G, Masciullo V, Bellacosa A, Benedetti-Panici P, Mancuso S. 1997. Cyclin E and cdk2 alterations in ovarian cancer: amplification and overexpression. Int.J. Cancer 74, 390–395.PubMedCrossRefGoogle Scholar
  8. 8.
    Kitamura T, Tange T, Terasawa T, Chiba S, Kuwaki T, Miyagawa K, Piao YF, Miyazono K, Urabe A, Takaku F. 1989. Establishment and characterization of a unique human cell line that proliferates dependently on GMCSF, IL-3, or erythropoietin. J.Cell.Physiol. 140, 632–634.CrossRefGoogle Scholar
  9. 9.
    Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY,Google Scholar
  10. 10.
    Wang TTY, Phang JM. 1995. Effects of estrogen on apoptotic pathways in human breast cancer cell line MCF-7. Cancer Res. 55, 2487–2489.PubMedGoogle Scholar
  11. 11.
    Chen D, Klebe RJ. 1993. Controls for validation of relative reverse transcription-polymerase chain reaction assays. PCR Methods and Applications 3, 127–129.PubMedGoogle Scholar
  12. 12.
    Freeman WM, Walker SJ, Vrana KE. 1999. Quantitative RT-PCR: pitfalls and potential. BioTechniques 26, 112–125.PubMedGoogle Scholar
  13. 13.
    Ali SA, Sarto I, Steinkasserer A. 1997. Production of PCR mimics for any semiquantitative PCR application. BioTechniques 6, 1060–1062.Google Scholar
  14. 14.
    Corey E, Corey MJ. 1998. Detection of disseminated prostate cells by reverse transcription-polymerase chain reaction (RT-PCR): technical and clinical aspects. Int J Cancer 77, 655–673.PubMedCrossRefGoogle Scholar
  15. 15.
    Medhurst AD, Harrison DC, Read SJ, Campbell CA, Robbins MJ, Pangalos MN. 2000. The use of TaqMan RT-PCR assays for semiquantitative analysis of gene expression in CNS tissues and disease models. J.Neurosci.Methods 98, 9–20.PubMedCrossRefGoogle Scholar
  16. 16.
    Gibson UEM, Heid CA, Williams PM. 1996. A novel method for real time quantitative RT-PCR. Genome Methods 6, 995–1001.Google Scholar
  17. 17.
    Ririe KM, Rasmussen RP, Wittwer CT. 2001. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Analytical Biochem. 245, 154–160.CrossRefGoogle Scholar
  18. 18.
    Pfeffer U, Fecarotta E, Vidali G. 1995. Efficient one-tube RT-PCR amplification of rare transcripts using short sequence-specific reverse transcription primers. BioTechniques 18, 204–206.PubMedGoogle Scholar
  19. 19.
    Lion T. 1996. Control genes in reverse transcriptase-polymerase chain reaction assays. Leukemia 10, 1527–1528.PubMedGoogle Scholar
  20. 20.
    Chelly J, Kaplan J-C, Maire P, Gautron S, Kahn A. 1988. Transcription of the dystrophin gene in human muscle and non-muscle tissues. Nature 858–860.Google Scholar
  21. 21.
    Morosetti R, Park DJ, Chumakov AM, Grillier I, Shiohara M, Gombart AF, Nakamaki T, Wenberg K, Koeffler H. P. 1997. A novel, myeloid transcription factor, C/EBPɛ, is upregulated during granulocytic, but not monocytic, differentiation. Blood 90, 2591–2600.PubMedGoogle Scholar
  22. 22.
    Foley KP, Leonard MW, Engel JD. 1993. Quantitation of RNA using the polymerase chain reaction. Trends in Genetics 9, 380–385.PubMedCrossRefGoogle Scholar
  23. 23.
    Souazé F, Ntodou-Thomé A, Tran CY, Rostène W, Forgez P. 1996. Quantitative RT-PCR: limits and accuracy. BioTechniques 21, 280–285.PubMedGoogle Scholar
  24. 24.
    Marone M, Scambia G, Bonanno G, Rutella S, De Ritis D, Guidi F, Mancuso S, Leone G, Pierelli L. 2001. Transforming growth factor-β1 transcriptionally activates CD34 and prevents induced differentiation of TF-1 cells in the absence of any cell cycle effects. Leukemia, in press Google Scholar

Copyright information

© Springer 2001

Authors and Affiliations

  • Maria Marone
    • 1
  • Simona Mozzetti
    • 1
  • Daniela De Ritis
    • 1
  • Luca Pierelli
    • 2
    • 1
  • Giovanni Scambia
    • 1
  1. 1.Department of GynecologyCatholic UniversityRomeItaly
  2. 2.Department of HematologyCatholic UniversityRomeItaly

Personalised recommendations