Development of a novel quantitative assay for the measurement of chloramphenicol acetyl transferase (CAT) mRNA.
Most host cells transfected with chloramphenicol acetyl transferase (CAT) expressing plasmids display relatively low levels of constitutive CAT activity. While this is ideal to study factors that enhance gene transcription, decreases in CAT levels are difficult to quantitate, using conventional CAT assays. Thus, investigators have used cell activating agents or co-transfection of the cell lines with a second enhancer plasmid to yield higher levels of CAT activity. However, such measures can interfere with the cellular pathways studied and eventually alter the results. To avoid this problem, our laboratory has designed an RT-PCR assay to quantitate CAT mRNA. The ability of this assay to detect CAT mRNA but not CAT DNA demonstrates its specificity and is achieved using a tailed oligoprimer for the reverse transcription step. This assay is able to measure the equivalent of as few as eight copies of CAT mRNA, is reproducible and relatively easy to perform. The quantitative capability of the assay relies on a constant production of CAT mRNA, which is achieved using permanently transfected and cloned cell lines bearing a defined number of CAT DNA copies per cell. This assay provides a tool for monitoring events at the transcriptional level and thereby complements the currently used CAT ELISA and thin-layer chromatography assays.
Knuchel
M
M
Department of Pathology, Emory University School of Medicine, Atlanta, GA 30322.
Bednarik
D P
DP
Chikkala
N
N
Villinger
F
F
Folks
T M
TM
Ansari
A A
AA
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014162:Transfection; D014773:Virology / Q000379:methods
D017931:DNA Primers; D012333:RNA, Messenger; D015500:Chloramphenicol O-Acetyltransferase