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) reporter 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 of the hGH cDNA and not the reporter plasmid. The assay is sensitive and simple to perform, requires no special equipment, and can quantify reporter cDNA concentration over a broad range.
Morales
M J
MJ
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Gottlieb
D I
DI
D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant; D005069:Evaluation Studies as Topic; D015870:Gene Expression; D013006:Growth Hormone / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012680:Sensitivity and Specificity; D014162:Transfection
D004274:DNA, Recombinant; D012333:RNA, Messenger; D013006:Growth Hormone; D004247:DNA