A PCR-based assay for reporter gene expression.
Transient transfection is a widely used tool for the identification of cis-acting regulatory elements. These elements are detected by their effect on the expression of a reporter gene, which is quantified by measuring the reporter gene product in the form of mRNA, protein (hGH), or enzymes (CAT, luciferase). Measurements of mRNA levels have several advantages over enzyme or protein assays. However, mRNA quantification by RNase protection or S1 mapping has considerably lower signal-to-background ratio than protein assays and is therefore less sensitive. In this paper we report the development of a system that takes advantage of the polymerase chain reaction (PCR) to quantify rabbit beta-globin reporter gene expression. Cells are co-transfected with constructs whose activity is to be tested and a reference plasmid with a small deletion in the second exon of the beta-globin gene. We show that the ratio of the two amplified cDNA signals is a highly reliable measure of test gene expression. The sensitivity of this assay is at least 1000-fold higher than RNase protection.
Sperisen
P
P
ISREC, Epalinges, Switzerland.
Wang
S M
SM
Reichenbach
P
P
Nabholz
M
M
D000818:Animals; D001483:Base Sequence; D002872:Chromosome Deletion; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D005914:Globins / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D012680:Sensitivity and Specificity; D014162:Transfection
D012333:RNA, Messenger; D005914:Globins; D004247:DNA