The reaction mixture consisted of 2 µL (or 5 ng) of total RNA, 0.5 µL of 10 × RT buffer, 1 µL of 5 × RT primer, 0.05 µL of dNTPs (100 mM), 0.06 µL of RNase Inhibitor (20 U/µL), and 0.33 µL of MultiScribe Reverse Transcriptase (50 U/µL) in a final reaction volume of 5 µL. The reaction mixture for real-time PCR consisted of 4 µL of a template cDNA, 10 µL of TaqMan Fast Universal PCR Master Mix (Applied Biosystems), and 1 µL of 20 × primer/probe mixture in a total reaction volume of 20 µL. Real-time RT-PCR was performed with precycling heat activation
at 95°C for 20 sec, followed Inhibitors,research,lifescience,medical by 40 cycles of denaturation at 95°C for 3 sec and annealing/extension at 60°C for 30 sec in an Applied neverless Biosystems 7500 Fast Real-Time PCR System. Susceptible to RNase degradation To evaluate the susceptibility Inhibitors,research,lifescience,medical to RNase, RNA extracted from HT-29 cells was treated using RNase (Qiagen, final concentration:
5 µg/mL) at 4°C or 37°C for 0, 5, 10, 20, and 30 min. After the treatment, all samples were electrophoresed using a Cosmo-I microcapillary electrophoresis, the concentrations of total RNA were evaluated using a NanoDrop UV spectrometer, and the expressions of miRNA from HT-29 cells were analyzed using real-time RT-PCR. selleck chemicals llc Analysis of RNA protection from RNase HT-29 cells (5 × 105 cells) were plated into a 10-cm cell Inhibitors,research,lifescience,medical culture plate (Corning, Corning, NY). After an exchange for 10 mL of fresh medium the next day, HT-29 cells were cultured for 48 hr. The HT-29 cells were then incubated Inhibitors,research,lifescience,medical at 37°C for 0, 30, 60, and 90 min after addition of RNase (final concentration, 5 µg/mL). The culture media and cells were processed as described above, and free miRNA, exosomal miRNA, and cellular miRNA could be obtained. Three replicates were performed in each sample. One gram of fecal sample from 3 volunteers was put into Stomacher Lab Blender Bags (Seward) and incubated at 37°C for 0, 30, 60, and 90 min after the addition of RNase (final concentration, 5 µg/mL). The fecal samples were processed, and fecal miRNA, exosomal miRNA, and colonocyte
miRNA could Inhibitors,research,lifescience,medical be obtained as described above. Statistical analysis The miRNA expression analyses were conducted using the comparative Batimastat Ct (threshold cycle) method. The relative quantification for each miRNA was analyzed using a two-sided t-test. Statistical analyses were performed using StatView Ver. 5 for Windows (Abacus Concepts, Berkeley, CA). P<0.05 was considered statistically significant. Results Degradation of naked RNA from HT-29 cells using RNase Total RNA extracted from HT-29 cells was treated, using 5 µg/mL of RNase, and electrophoresed. Two peaks, 18S and 28S ribosomal RNA (rRNA), were observed in the total RNA without treatment of RNase (Fig 1A). On the other hand, no rRNA peak was observed in the total RNA treated with RNase. Small RNAs, including miRNA or degrading RNA, were observed in all samples.