![]() ![]() 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. ![]() ![]() 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). Total RNA was isolated and quantified using the RiboGree&ncircledR fluorescent dye with the VersaFluor Fluorometer System. ![]() 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. This approach was evaluated by using the beta-actin gene as a reference molecule for measuring of mRNA stability. BACKGROUND: We describe an alternative method to determine mRNA half-life (t1/2) based on the Real-Time RT-PCR procedure. ![]()
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