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Annals of Clinical & Laboratory Science 34:47-56 (2004)
© 2004 Association of Clinical Scientists

Quantification of Human PPAR{gamma}1 Gene Expression by Competitive PCR Using an Homologous Internal Standard

Nik Soriani Yaacob1, Ruzilawati Abu Bakar1 and Mohd-Nor Norazmi2
Schools of 1 Medicine and 2 Health Sciences, Universiti Sains Malaysia, Kelantan, Malaysia

Address correspondence to Nik Soriani Yaacob Ph.D., Department of Chemical Pathology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia; tel 609 766 4750; fax 609 765 3370; e-mail soriani{at}kb.usm.my.

The polymerase chain reaction (PCR) is useful for amplifying specific mRNAs, particularly those present in low copy numbers. However, due to the exponential nature of the amplification process, PCR cannot readily be used to quantify gene expression. A competitive PCR technique was developed to address this shortcoming. An internal standard that is 100% homologous to, but shorter than, the target gene was constructed. The practicality of the method was demonstrated by determining the expression levels of a human transcription factor, peroxisome proliferator-activated receptor gamma 1 (hPPAR{gamma}1) which is normally present in low copy numbers in selected cells. A mock system was used to test the accuracy and sensitivity of the method, which was subsequently used to determine the expression of this receptor in lipopolysaccharide (LPS)-activated monocytes, which are known to express hPPAR{gamma}1 differentially during cellular activation. Densitometric analysis showed that the competitive PCR method reliably estimated the expression levels of hPPAR{gamma}1 at the attomole (10-18) level in monocytes.

Keywords: competitive PCR, monocyte, PPAR{gamma}1




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