Real-time RT-PCR Analysis of MRNA Decay: Half-life of Beta-actin MRNA in Human Leukemia CCRF-CEM and Nalm-6 Cell Lines

Overview

Journal Cancer Cell Int

Publisher Biomed Central

Date 2002 May 4

PMID 11988110

Citations 40

Authors

Guy J Leclerc

Gilles M Leclerc

Julio C Barredo

Affiliations

Soon will be listed here.

Abstract

BACKGROUND: We describe an alternative method to determine mRNA half-life (t1/2) based on the Real-Time RT-PCR procedure. This approach was evaluated by using the beta-actin gene as a reference molecule for measuring of mRNA stability. RESULTS: Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGree&ncircledR; fluorescent dye with the VersaFluor Fluorometer System. One &mgr;g of total RNA was reverse transcribed and used as template for the amplification of a region of the beta-actin gene (231 bp). To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, beta-actin mRNAs were quantified by Real-Time RT-PCR using the SYB&RcircledR; Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the beta-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t1/2 value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h). CONCLUSIONS: We have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that beta-actin mRNA half-life can be affected by the cellular growth rate.

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