Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines.
Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines.
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DOI：
10.1186/1475-2867-2-1
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发表时间：
2002-03-07
影响因子：
5.8
通讯作者：
Barredo JC
中科院分区：
文献类型：
--
作者：
Leclerc GJ;Leclerc GM;Barredo JC
我们描述了一种替代方法，以确定mRNA半衰期（t1/2）的基础上实时RT-PCR程序。通过使用β-肌动蛋白基因作为用于测量mRNA稳定性的参考分子来评估该方法。用各种浓度的放线菌素D处理人白血病Nalm-6和CCRF-CEM细胞以阻断转录，并定期取出等分试样。使用RiboGreen®荧光染料和VersaFluor荧光计系统分离和定量总RNA。逆转录Iyg总RNA并用作模板用于扩增β-肌动蛋白基因的区域（231 bp）。为了产生标准曲线，使用含有cDNA扩增片段的pBactin-231载体的连续十倍稀释液，使用SYBR®绿色I荧光染料通过实时RT-PCR定量β-肌动蛋白mRNA，并使用iCycle iQ系统软件分析数据。使用这种方法，β-actin mRNA在Nalm-6和CCRF-CEM细胞中分别显示出6.6 h和13.5 h的半衰期。获得的Nalm-6的t1/2值与使用正常人白细胞（5.5 h）的北方印迹研究估计的值相当。我们已经开发了一种快速，灵敏，可靠的方法，基于实时RT-PCR测量mRNA半衰期。我们的结果证实，β-肌动蛋白mRNA的半衰期可以受到细胞生长速率的影响。
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.