On the other hand, since the expression level of the oncogenic miRNAs, such as miR-17-92 cluster, miR-21, and miR-135, in cancer tissue was higher than in normal tissue, these oncogenic miRNAs could be used for a marker of prognosis or poor response to chemotherapy (9)-(14). Exosomes are nanoparticles, 50-100 nm in diameter,

and are released from cells into extracellular matrixes through fusion of multivesicular bodies with the plasma membrane (15),(16). Recent reports indicate that miRNAs are circulating stably in bloodstream wrapping in exosomes, which can prevent Inhibitors,research,lifescience,medical RNase from Pfizer Licensed Compound Library datasheet degrading the miRNAs (17)-(21). Therefore several methods for miRNA-based early cancer detection Inhibitors,research,lifescience,medical using serum, plasma, and urine are reported (21)-(23). Also, several studies are available of the possible use of the miRNA-based method for CRC screening in serum (24),(25) and in feces (26). We have been developing new screening methods for CRC by applying molecular biological tools to exfoliated colonocytes isolated from naturally evacuated feces (27)-(29). In the past few years especially, we have reported the fecal RNA test, including the CRC-related gene expression analysis (30) and the CRC-related miRNA expression analysis (31). Within this context, we investigate the stability of miRNA in feces. Materials and Methods Cell line and fecal samples The human colorectal

Inhibitors,research,lifescience,medical cancer cell line HT-29 (American Tissue Culture Collection, Rockville,

MD) was cultured in the Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin G, 100 µg/mL streptomycin, Inhibitors,research,lifescience,medical and 0.25 µg/mL amphotericin B at 37°C in a humidified atmosphere of 5% CO2: 95% air. Naturally evacuated fecal samples were obtained from 3 healthy volunteers with no endoscopical abnormalities. Volunteers were instructed to evacuate at home into a disposable 5 × 10-cm polystyrene tray (AsOne, Osaka, Japan) and to bring the fecal sample to our laboratory at 4°C. The samples were then immediately prepared for the next step. Isolation of exfoliated colonocytes Inhibitors,research,lifescience,medical from feces using EpCAM beads EpCAM (epithelial cell adhesion molecule) 17-DMAG (Alvespimycin) HCl beads (JSR, Tsukuba, Japan), immunomagnetic beads conjugated with EpCAM monoclonal antibody (mAb) (clone B8-4), were used for isolation of colonocyte from feces (32). Fecal samples were processed as described previously (28). Briefly, one gram of fecal sample was homogenized with a buffer (40 mL) consisting of Hanks’ solution, 10% fetal bovine serum (FBS), and 25 mM HEPES buffer (pH 7.35) at 200 rpm for 1 min using a Stomacher system (Seward, Thetford, UK). The homogenate was filtered through a nylon filter (pore size, 512 µm), and following the addition of 40 µL of EpCAM beads, the sample mixture was incubated for 30 min under gentle rolling conditions at room temperature.