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A polymerase chain reaction-based method for detection and quantification of reporter gene expression in transient transfection assays






Analytical Biochemistry 210(1): 188-194

A polymerase chain reaction-based method for detection and quantification of reporter gene expression in transient transfection assays

Transcription in higher eukaryotes is often studied by the use of transient transfection assays. These experiments are performed by cloning putative cis-acting transcriptional elements (i.e., a promoter or enhancer) with a reporter gene that codes for a protein not expressed by the target cells. Although this approach is useful in many cases, the limited sensitivity of reporter assays can prevent studies in cases where few cells are obtainable or efficiency of transfection is low. We present an alternative approach. Cells are transfected with a plasmid containing a promoter with a human growth hormone (hGH) receptor gene. After an incubation period, RNA is isolated, and DNA complementary to the growth hormone mRNA is produced. The reporter cDNA concentration is measured by quantitative polymerase chain reaction (PCR). We have designed PCR primers that span the mRNA splice sites of the human growth hormone gene; these ensure exclusive amplification on the hGH cDNA and not the reporter plasmid. The assay is sensitive and simple to perform, requires no special equipment, and can quantify receptor cDNA concentration over a broad range.

Accession: 002287032

PMID: 8489016

DOI: 10.1006/abio.1993.1171

Download PDF Full Text: A polymerase chain reaction-based method for detection and quantification of reporter gene expression in transient transfection assays



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