Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines
© Leclerc et al; licensee BioMed Central Ltd. 2002
Received: 28 November 2001
Accepted: 07 March 2002
Published: 07 March 2002
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 β-actin gene as a reference molecule for measuring of mRNA stability.
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 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).
We 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.
Determination of mRNA half-life is important to our understanding of gene expression and mechanisms involved in the regulation of the level of transcripts in response to environmental changes or developmental cues. In addition, the stability of mRNA may determine how rapidly the synthesis of the encoded protein can be shut down after transcription ceases. mRNA half-life can be determined by densitometric analysis of in situ hybridization histochemistry  or by Northern blot analysis  of RNA samples removed from cells treated with transcriptional inhibitors such as actinomycin D (ActD) , α-amanitin , or 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) . Although reliable, these multi-step methods are laborious and time-consuming. The advent of new technologies such as the Real-Time PCR allows rapid and exact measurement of copy number of molecules present in the sample . Real-Time Reverse Transcriptase PCR (RT-PCR) allows precise and reproducible quantitative determination of the number of mRNA transcripts synthesized [7, 8]. We have developed a rapid and reliable Real-Time 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.
To evaluate that approach, the β-actin gene was used as a reference molecule for mRNA stability. Actin proteins are components of the microfilament which play a crucial role in maintaining cell shape and motility. Expression of β-actin has been shown to be relatively constant as cells progress through the cell cycle  and has been used as a standard for an unchanging protein and mRNA in studies of gene regulation. In this study, we used the human leukemia cell lines CCRF-CEM (T-cell lineage, Acute Lymphoblastic Leukemia (ALL)) and Nalm-6 (B-cell precursor, ALL) that respond differently to antifolate drugs, such as methotrexate (MTX). Nalm-6 cells were shown to be more sensitive to MTX when compared to CCRF-CEM .
Results and Discussion
In summary, we described an alternative method using Real-Time RT-PCR to determine the rate of mRNA degradation by accurately measuring the number of mRNA molecules relative to total RNA. Using this approach, we obtained a values for the β-actin mRNA half-life in Nalm-6 cells that are comparable to those estimated from Northern blot studies using normal human leukocytes . Therefore, Real-Time RT-PCR is a reliable method for measuring mRNA half-life. Because of its sensitivity, half-life of mRNAs expressed at very low level can be determined in cases in which Northern blots may not be sensitive enough. The length of the amplified fragment is important to determine the mRNA half-life because incomplete or degraded mRNA can interfere with the measurement of the actual mRNA half-life. Ideally, full-length cDNA molecules should be amplified to ensure integrity and identity of the mRNA species. The use of the fluorogenic SYBR Green I dye limits the length of the amplified product (cDNA recommended to be less than 200 bp). However, the use of TaqMan® (Applied Biosystems, Foster City, CA, USA), molecular beacons (Molecular Probes, Inc., Eugene, OR, USA), or fluorescence resonance energy transfer (FRET) probes (Roche Molecular Biochemicals, Indianapolis, IN, USA) could overcome this limitation and allow amplification of longer PCR products. In addition, since multiplex Real-Time RT-PCR can be achieved in the same reaction tube using different fluorogenic dyes, this method could be modified to simultaneously estimate mRNA half-life of several genes. Thus, our approach represents a rapid and sensitive assay to determine mRNA half-life.
Materials and Methods
Leukemia cell Lines
The human leukemia cell lines CCRF-CEM (T-cell, ALL) and Nalm-6 (B-cell precursor, ALL) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and DSMZ (Braunschweig, Germany), respectively. Both cell lines were grown in RPMI 1640 (Sigma-Genosys, Woodlands, TX, USA) supplemented with 10% fetal bovine serum at 37°C under a 5% CO2 atmosphere. Culture medium was changed according to standard tissue culture techniques to insure cellular integrity. Trypan blue exclusion methodology was used to assess cell viability.
RNA isolation and Real Time RT-PCR
Total RNA was isolated using the RNeasy kit (Qiagen, Inc., Valencia, CA, USA) and its concentration determined using the RiboGreen® fluorescent dye (Molecular Probes, Inc., Eugene, OR, USA) with the VersaFluor Fluorometer System (BioRad, Hercules, CA, USA). Quality and integrity of total RNA was assessed on 1% formaldehyde-agarose gels. First-strand cDNA was synthesized using 1 μg of total RNA (DNase-treated) in a 20 μl reverse transcriptase reaction mixture as described by Leclerc and Barredo . A region of the β-actin mRNA was amplified using primers BA67 (5'-GCGGGAAATCGTGCGTGACATT) and BA68 (5'-GATGGAGTTGAA GGTAGTTTCGTG), as described by Lenz et al. . The cDNA amplified fragment (231 bp) was cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA) to generate the plasmid pBactin-231 (4139 bp). Serial ten-fold dilutions (104 to 109 molecules) of pBactin-231 were used as a reference molecule for the standard curve calculation (Figure 2). All Real-Time PCR reactions were performed in a 25 μl mixture containing 1/20 volume of cDNA preparation (1 μl), 1X SYBR Green buffer (PE Applied Biosystems, Foster City, CA, USA), 4 mM MgCl2, 0.2 μM of each primers (BA67 and BA68), 0.2 mM dNTPs mix and 0.025 Unit of AmpliTaq Gold® thermostable DNA polymerase (Applied Biosystems, Foster City, CA, USA). Real-Time quantitations were performed using the BIO-RAD iCycler iQ system (BioRad, Hercules, CA, USA). The fluorescence threshold value was calculated using the iCycle iQ system software.
The authors would like to thank Florence B. Leclerc and Sanja Altman-Hamamdzic for helpful discussion, and the Medical University of South Carolina, Biotechnology Resource Laboratory, Charleston, SC, for their assistance with DNA sequence analysis. This work was supported in part by research Grant NIH/NCI CA72734 (to J. C. B.) and the Jolly Foundation Award (to G. J. L. and J. C. B.) and by the Monica Kreber Golf Classic, Charleston, SC.
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