Quantification of human PPARgamma1 gene expression by competitive PCR using an homologous internal standard.
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 (hPPARgamma1) 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 hPPARgamma1 differentially during cellular activation. Densitometric analysis showed that the competitive PCR method reliably estimated the expression levels of hPPARgamma1 at the attomole (10(-18)) level in monocytes.
Yaacob
Nik Soriani
NS
Department of Chemical Pathology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. soriani@kb.usm.my
Bakar
Ruzilawati Abu
RA
Norazmi
Mohd-Nor
MN
D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D009000:Monocytes / Q000737:chemistry; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D012015:Reference Standards; D017385:Sequence Homology; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D015854:Up-Regulation
D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors