Skip to main content

A Stochastic PCR Approach for RNA Quantification in Multiple Samples

  • Protocol

Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 26))

Abstract

When studying the effect of various treatments on gene expression in humans, one occasionally is faced with the problem of detecting small changes in transcript levels in minute tissue samples. In addition, interindividual variations can be quite large and may even be the major source of variation (1). Therefore, numerous samples usually have to be analyzed to detect such small variations in gene expression.

This is a preview of subscription content, log in via an institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Gundersen, H. J. G and Osterby, R. (1980) Optimizing sampling efficiency of stereological studies in biology: or ‘Do more less well!’ J. Microscopy 121, 65–73.

    Google Scholar 

  2. Gilliland, G, Perin, S., Blanchard, K., and Bunn, H. F. (1990) Analysis of cytokine mRNA and DNA: Detection and quantitation by competitive polymerase chain reaction. Proc. Natl. Acad. Sci. USA 87, 2725–2729.

    Article  CAS  PubMed  Google Scholar 

  3. Wiesner, R. J., Ruegg, J. C, and Morano, I. (1992) Counting target molecules by exponential polymerase chain reaction: Copy number of mitochondrial DNA in rat tissues. Biochem. Biophys. Res. Comm. 183, 553–559.

    Article  CAS  PubMed  Google Scholar 

  4. Wang, A., Doyle, M. V., and Mark, D. F. (1989) Quantitation of mRNA by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 86, 9717–9721.

    Article  CAS  PubMed  Google Scholar 

  5. Chelly, A., Montarras, D., Pinset, C., Berwald-Netter, Y., and Kaplan, I.-C. (1990) Quantitative estimation of minor mRNAs by cDNA-polymerase chain reaction: application to dystrophin mRNA in cultured myogenic and brain cells. Eur. J. Biochem. 187, 691–698.

    Article  CAS  PubMed  Google Scholar 

  6. Ferre, F. (1993) Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods Applic. 2, 1–9.

    Google Scholar 

  7. Clementi, M., Menzo, S., Bagnarelli, P., Manzin, A., Valenza, A., and Varaldo, P. E. (1993) Quantitative PCR and RT-PCR in virology. PCR Methods Applic. 2, 191–196.

    CAS  Google Scholar 

  8. Reischl, U. and Kochanowski, B. (1995) Quantitative PCR. Mol. Biotech. 3, 55–71.

    Article  CAS  Google Scholar 

  9. Nedelman, J., Haegerty, P., and Lawrence, C. (1992) Quantitative PCR with internal controls. Comput. Applic. Biosci. 8, 65–70.

    CAS  Google Scholar 

  10. Becker-Andre, M. and Hahlbrok, K. (1989) Absolute mRNA quantification using the polymerase chain reaction (PCR): a novel approach by a PCR aided transcript titration assay (PATTY). Nucleic Acid Res. 17, 9437–9446.

    Article  CAS  PubMed  Google Scholar 

  11. Murphy, L. D., Herzog, C. E., Rudick, J. B., Fojo, A. T., and Bates, S. E. (1990) Use of the polymerase chain reaction in the quantitation of mdr-1 gene expression. Biochemistry 29, 10,351–10,356.

    Article  CAS  PubMed  Google Scholar 

  12. Noonan, K. E., Beck, C., Holzmayer, T. A., Chin, J. E., Wunder, J. S., Andrulis, I. l., Gazdar, A. F., Willman, C. L., Griffith, B., Von Hoff, D. D., and Roninson, I. B. (1990) Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction. Proc. Natl. Acad. Sci. USA 87, 7160–7164.

    Article  CAS  PubMed  Google Scholar 

  13. Hoof, T., Riordan, J. R., and Tuemmler, B. (1991) Quantitation of mRNA by the kinetic polymerase chain reaction assay: a tool for monitoring P-glycoprotein gene expression. Anal. Biochem. 196, 161–169.

    Article  CAS  PubMed  Google Scholar 

  14. Chelly, J., Kaplan, J.-C., Maire, S., Gautron, S., and Kahn, A. (1988) Transcription of the dystrophin gene in human muscle and non-muscle tissues. Nature 333, 858–860.

    Article  CAS  PubMed  Google Scholar 

  15. Rappole, D. A., Wang, A., Mark, D., and Werb, Z. (1989) Novel method for studying mRNA phenotypes in single or small numbers of cells. J. Cell. Biochem. 39, 1–11.

    Article  Google Scholar 

  16. Puntschart, A., Jostarndt, K., Hoppeler, H., and Billeter, R. (1994) An efficient polymerase chain reaction approach for the quantitation of multiple RNAs in human tissue samples. PCR Methods Applic. 3, 232–238.

    CAS  Google Scholar 

  17. Puntschart, A., Claassen, H., Jostarndt, K., Hoppeler, H., and Billeter, R. (1995) mRNAs of enzymes involved in energy metabolism and mtDNA are increased in endurance-trained athletes. Am. J. Physiol. 269, C619–C625.

    CAS  PubMed  Google Scholar 

  18. Yang, B., Yolken, R., and Viscidi, R. (1993) Quantitative polymerase chain reaction by monitoring enzymatic activity of DNA polymerase. Anal. Biochem. 208, 110–116.

    Article  CAS  PubMed  Google Scholar 

  19. Ito, H., Miller, S. C., Akimoto, H., Torti, S. V., Taylor, A., Billingham, M. E., and Torti, F. M. (1991) Evaluation of mRNA levels by the polymerase chain reaction in small cardiac tissue samples. J. Mol. Cell. Cardiol. 23, 1117–1125.

    Article  CAS  PubMed  Google Scholar 

  20. Dieffenbach, C. W. and Dveksler, G. S. (1995) PCR Primer: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  21. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.

    Article  CAS  PubMed  Google Scholar 

  22. Sambrook, J., Fritsch, E. F., and Maniatis, T. (eds.) (1989) Molecular Cloning. A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 1999 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Puntschart, A., Vogt, M. (1999). A Stochastic PCR Approach for RNA Quantification in Multiple Samples. In: Kochanowski, B., Reischl, U. (eds) Quantitative PCR Protocols. Methods in Molecular Medicine™, vol 26. Humana Press. https://doi.org/10.1385/0-89603-518-2:277

Download citation

  • DOI: https://doi.org/10.1385/0-89603-518-2:277

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-518-8

  • Online ISBN: 978-1-59259-262-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics