Abstract
BackgroundWe 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 β-actin gene as a reference molecule for measuring of mRNA stability.ResultsHuman 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 RiboGreen® fluorescent dye with the VersaFluor Fluorometer System. One μg of total RNA was reverse transcribed and used as template for the amplification of a region of the β-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, β-actin mRNAs were quantified by Real-Time RT-PCR using the SYBR® Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the β-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).ConclusionsWe have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that β-actin mRNA half-life can be affected by the cellular growth rate.
Highlights
We describe an alternative method to determine mRNA half-life (t1/2) based on the Real-Time RT-PCR procedure
The β-actin mRNA half-life was determined from CCRFCEM and Nalm-6 cell lines treated with 0.5 μM, 1 μM, and 5 μM actinomycin D (ActD) (Sigma-Genosys, Woodlands, TX, USA)
It has been shown that ActD inhibits cell proliferation by forming a stable complex with single-stranded DNA and blocking the movement of RNA polymerase that interferes with DNA-dependent RNA synthesis [11]
Summary
We describe an alternative method to determine mRNA half-life (t1/2) based on the Real-Time RT-PCR procedure. MRNA half-life can be determined by densitometric analysis of in situ hybridization histochemistry [1] or by Northern blot analysis [2] of RNA samples removed from cells treated with transcriptional inhibitors such as actinomycin D (ActD) [3], α-amanitin [4], or 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) [5]. Reliable, these multi-step methods are laborious and time-consuming. We have developed a rapid and reliable RealTime quantitative RT-PCR approach to determine mRNA half-life based on the SYBR® Green I fluorogenic dye (Molecular Probes, Inc., Eugene, OR, USA) and relative to the amount of total RNA per cell samples
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