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Avoidance of False Positives in PCR-Based mRNA Differential Display During Investigation of Nonstandardized Tissues or Cells

¹ Inst. für Arterioskleroseforsch. an der Univ. Münster, Domagkstr. 3, 48149 Münster, and
² Inst. für Klin. Chem. und Laboratoriumsmed., Univ. Münster, Albert-Schweitzer-Str. 33, 48149 Münster, Germany;
^a author for correspondence: fax 49-251-83 6208, e-mail cullen@uni-muenster.de

Although not yet a part of clinical chemical practice, analyzing patterns of differential gene expression in tissues such as cancers will likely become a routine method in coming years. The technique of PCR-based differential display developed by Liang and Pardee (1) has become increasingly popular during the last 4 years and is a considerable advance on conventional methods used to identify differentially expressed genes, such as differential hybridization and subtractive library construction. The advantages of differential display are the low RNA requirement and high versatility. However, serious problems remain, one of the most important being the high rate of false positives. Several methods have been described to deal with this problem, including the simultaneous display of PCR products from two uninduced and two induced lines with the requirement that the patterns from the pairs of uninduced or induced lines agree (2); the running of duplicate, identical samples from each RNA preparation side by side (3)(4); the display of PCR products from uninduced and induced lines over a time course of induction (2); and the repeating of experiments for those lanes in which putative candidate bands were identified (5).

We describe here a method for effectively eliminating false positives and spurious true positives (i.e., genes that really are regulated but whose regulation is not a result of the manipulation under investigation). This method is particularly useful when investigating gene expression in nonstandardized material such as fresh human or animal tissue or cells.

We use PCR-based differential display to investigate gene regulation in human foam cells produced by loading macrophages for 48 h with 80 µg/mL acetlyated (Ac) or oxidized (Ox) low-density lipoprotein (LDL). Human peripheral blood monocytes are isolated from volunteers by a combination of cell separation and countercurrent . . . [Full Text of this Article]

Acknowledgments

References

The following articles in journals at HighWire Press have cited this article:

				S. Brandt, S. Kloska, T. Altmann, and J. Kehr Using array hybridization to monitor gene expression at the single cell level J. Exp. Bot., December 1, 2002; 53(379): 2315 - 2323. [Abstract] [Full Text] [PDF]

				J. Sturtevant Applications of Differential-Display Reverse Transcription-PCR to Molecular Pathogenesis and Medical Mycology Clin. Microbiol. Rev., July 1, 2000; 13(3): 408 - 427. [Abstract] [Full Text] [PDF]