Nucleic acid stain 1

Total RNA was isolated from cells at indicated time points using Trizol (Omega Bio-tek, Shenzhen, China), according to the manufacturer’s instructions, and then reverse transcribed into cDNA with M-MLV Reverse Transcriptase (Promega, Fitchburg, WI, USA). The cDNA was analyzed by RT-PCR using rTaq DNA polymerase (Takara, Tokyo, Japan). Amplicons from the RT-PCR reactions were separated on a 1.5% agarose gel, which was stained with nucleic acid stain I (Roche Diagnostics, Mannheim, Germany), photographed, and analyzed using the Gene Tools Analysis Software (Syngene, Cambridge, UK). The cDNA was performed by qRT-PCR using a LightCycler 96 instrument (Roche). The qRT-PCR data were presented as normalized ratios, which were calculated using ΔΔCT method by LightCycler system and software (Roche, Basel, Switzerland). The human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard. The primer sequences are listed in Table 1.

Total RNA was extracted from SW480, DLD-1 and HT29 cells using Trizol (Bio Basic Inc., Canada) according to the manufacturer’s instructions. First-strand cDNA synthesis was carried out with 2 µg RNA from each cell line and Moloney murine leukemia virus RT (MMLV-RT; MBI Fermentas, Amherst, NY, USA), then amplified in a 50-µl reaction mixture containing 50 mM of each of the four dNTPs, 3 U of Taq DNA polymerase (Promega), and 0.5 mM of primers (Sangon Co., Shanghai, China). The primers 5′-GCTGCTCAGGTCTTTCTTTTATG-3′ and 5′-CGACGTAGTCCATGTTCACAA-3′ were used to amplify a 252-bp region within the hTERT gene. As an internal control, expression of glyceraldehyde phosphate dehydrogenase (GAPDH) was assayed using the primers 5′-CTCAGACACCATGGGGAAGGTGA-3′ and 5′-ATGATCTTGAGGCTGTTGTCATA-3′ to amplify a 450-bp region. Amplification reactions were performed for 30 cycles of 94°C for 30 s, 56°C for 45 s and 72°C for 45 s. PCR products were separated on a 2.0% agarose gel, which was stained with nucleic acid stain I (Roche Diagnostics, Mannheim, Germany), photographed and analyzed semi-quantitatively using GeneTools Analysis Software (Syngene, Cambridge, UK). The cell line showing the highest level of hTERT mRNA was chosen for further study.