Development of a novel quantitative assay for the measurement of chloramphenicol acetyl transferase (CAT) mRNA

Marlyse Knuchel, Daniel P. Bednarik, Nathaniel Chikkala, Francois Villinger, Thomas M. Folks, Aftab A. Ansari

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)325-338
Number of pages14
JournalJournal of Virological Methods
Volume48
Issue number2-3
DOIs
StatePublished - 1994
Externally publishedYes

Fingerprint

Chloramphenicol
Transferases
Messenger RNA
Plasmids
Cell Line
DNA
Thin Layer Chromatography
Reverse Transcription
Transfection
Enzyme-Linked Immunosorbent Assay
Research Personnel

Keywords

  • CAT assay
  • PCR
  • Tailed RT-PCR

ASJC Scopus subject areas

  • Virology

Cite this

Development of a novel quantitative assay for the measurement of chloramphenicol acetyl transferase (CAT) mRNA. / Knuchel, Marlyse; Bednarik, Daniel P.; Chikkala, Nathaniel; Villinger, Francois; Folks, Thomas M.; Ansari, Aftab A.

In: Journal of Virological Methods, Vol. 48, No. 2-3, 1994, p. 325-338.

Research output: Contribution to journalArticle

Knuchel, Marlyse ; Bednarik, Daniel P. ; Chikkala, Nathaniel ; Villinger, Francois ; Folks, Thomas M. ; Ansari, Aftab A. / Development of a novel quantitative assay for the measurement of chloramphenicol acetyl transferase (CAT) mRNA. In: Journal of Virological Methods. 1994 ; Vol. 48, No. 2-3. pp. 325-338.
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