Vic dye

Transcriptome candidates were verified by real time PCR using experiments following the nomenclature and description recommended by the MIQE Guidelines [75 (link)]. cDNA was generated from isolated RNA using the Superscript III Reverse Transcriptase Kit (Thermo Fischer Scientific, Waltham, MA). The hydrolysis probe labeled with FAM dye for Pde4b was purchased from Applied Biosystems. A GAPDH probe labeled with VIC dye (Applied Biosystems, Foster City, CA) was used as a reference gene. Normal colonic epithelial samples of both tumor-bearing and tumor-free Apc^Min/+ animals were analyzed for all array samples. The normal colonic epithelium of Apc^+/+ and tumor samples from Apc^Min/+ were included for additional quantitative comparisons of transcript levels. Additional RNA was obtained using the protocol described above (Qiagen). Each sample was run in triplicate and technical error between replicates did not exceed 5%. Fold-change expression was determined by calculating 2ⁿ for each sample, where n equals the difference in amplification cycle between the GAPDH reference and the test probe. The parameter deltaCT gives the number rounds of amplification needed to reach threshold compared to the number required for the control GAPDH probe.

Total cytoplasmic RNA was obtained from human lymphocytes by using the acid guanidinium thiocyanate phenol method. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay [25 (link)–28 (link)] of A₁, A_2A, A_2B, and A₃ ARs messenger RNAs (mRNAs) was performed using gene-specific fluorescently labeled TaqMan MGB Probe (minor groove binder) in an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Real-time RT-PCR for A₁, A_2A, A_2B, and A₃ ARs was carried out with the Assays-on-Demand TM Gene expression Products NM_000674, NM_000675, NM_000676, and NM_000677 (Applied Biosystems), respectively. For the real-time RT-PCR of the reference gene, the endogenous control human β-actin was used, and the probe was fluorescently labeled with VIC™ dye (Applied Biosystems).

Cells were cultured to 80–90% confluence. Total RNA was prepared using the Qiagen RNeasy Mini kit (Qiagen). The RT reaction was performed using 1 μg total RNA which was reverse-transcribed into cDNA using a random hexamer primer (GeneAmp RNA PCR Core kit; Applied Biosystems Life Technologies, Foster City, CA, USA), according to the manufacturer’s instructions. cDNA of the 7 selected genes and an internal reference gene (GAPDH) was produced from each sample and was quantified using a fluorescence-based real-time detection method (iCycler; Bio-Rad Laboratories, Inc., Hercules, CA, USA). RT-qPCR analysis was performed using the standard methods recommended by the RT-qPCR kit supplier (SYBR^® Green Dye-Based Gene Expression Detection; Applied Biosystems Life Technologies). Primer sequences used for detection of RXRα-regulated genes are shown in Table I (Cosmo Genetech, Seoul, Korea). For the endogenous control, human GAPDH labeled with VIC™ dye provided by Applied Biosystems Life Technologies was used. The amplification conditions were as follows: 30 sec at 95°C and 3 min at 95°C, and 30 sec at 95°C and 60 sec at 65°C for 40 cycles, followed by a final extension for 20 min at 72°C. The ratio between the values obtained provided the relative gene expression levels.