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Quantitation of Gene Expression by RT-PCR and HPLC Analysis of PCR Products

  • Franz Bachmair
  • Christian G. Huber
  • Guenter Daxenbichler
Part of the Methods in Molecular Biology book series (MIMB, volume 193)

Abstract

The quantitative measurement of specific mRNA species is of major importance for studies on gene expression. Northern blotting is a relatively insensitive method requiring microgram amounts of RNA. It is time consuming and semi-quantitative at best. Because of the limitations of Northern blotting, various strategies have been developed for quantitation of cDNA by polymerase chain reaction (PCR)-based methods (1, 2, 3, 4). Competitive PCR, in which a synthetic segment is co-amplified together with the target segment, is one of these approaches. Besides adding more steps to the experimental protocol, competitive PCR is limited because the competitor and target sequences are not necessarily amplified with the same efficiency, which impairs reliable quantitation (5). Recently, real-time PCR, in which the generated PCR-products are quantified fluorimetrically after each cycle, has become widely used (6). However, determination of fragment size for positive fragment identification is not—at least not directly—possible with this method.

Keywords

Polymerase Chain Reaction Product Retinoic Acid Receptor Guanidinium Thiocyanate Competitive Polymerase Chain Reaction Triethylammonium Acetate 
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.

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Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Franz Bachmair
    • 1
  • Christian G. Huber
    • 2
  • Guenter Daxenbichler
    • 3
  1. 1.Department of Obstetrics and GynecologyUniversity Hospital, University of InnsbruckAustria
  2. 2.Institute of Analytical Chemistry and RadiochemistryUniversity of InnsbruckAustria
  3. 3.Department of Obstetrics and GynecologyUniversity Hospital, University of InnsbruckAustria

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