Mir 675 3p

Total RNA was extracted using the commercially available illustraRNAspin Mini Isolation Kit (GE Healthcare, Italy), according to manufacturer's instructions. Tissue RNAs was extracted using the commercially available PureLink FFPE Total RNA Isolation Kit (Invitrogen cat.number 45-7015). Human colorectal cancer specimens (n = 22) were collected from the pathology archives of the Human Pathology Section, Ospedali Riuniti Villa Sofia-Cervello (Palermo) in accordance with the Declaration of Helsinki and with the policy of the Institute. RNA was reverse-transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, USA). Real-time PCR was performed in duplicates for each data point, and the oligonucleotides used are described in Table 1.
Changes in the target mRNA content relative to housekeeping gene (β-actin) were determined with the ΔΔct Method. For miRNA expression, 250 ng of RNA was reverse transcripted according to manufacturer's instructions (cat.number 4366596, TaqManMicroRNA Reverse Transcription, Applied Biosystem). Taqman probes were used to analyse: miR-675-5p (cat.number 4440887, Applied Biosystem), miR-675-3p (cat.number 4427975, Applied Biosystem), U6 (cat.number 4427975 Applied Biosystem), and H19 (Hs00262142_g1 Life Technologies). Changes in the target miRNA content relative to housekeeping U6 were determined with the ΔΔct Method.