Nucleospin microrna isolation kit

Blood samples were collected in the early morning within 24 hours after admission, centrifuged at 3000 g for 15 min at 4°C, and the obtained serum was stored at −80°C. Circulating miRs were isolated from 300 μL serum by using a NucleoSpin microRNA isolation kit (TaKaRa, Otsu, Japan).
Serum carboxy-terminal telopeptide of type I collagen (I-CTP) concentrations were determined by radioimmunoassay (Orion Diagnostica, Finland) [17 (link)]. Serum procollagen type III N-terminal propeptide (P3NP) levels were measured with enzyme-linked immunosorbent assay (ELISA) kit (MyBioSource, San Diego, CA, USA).

MiRNA was isolated from cultured cells by using a NucleoSpin microRNA isolation kit (Takara Bio Inc., Shiga, Japan). The relative expression levels were calculated by the ΔΔC_t method. Each value of ΔΔC_t was determined by use of a Thermal Cycler Dice Real Time System II model TP900/960 or TP870 (Takara Bio Inc.). The primers for PTBP1, PKM1, PKM2, and GAPDH were the following: PTBP1-sense, 5′-ATCAGGCCTTCATCGAGATGCACA-3′, and PTBP1-antisense, 5′-TGTCTTGAGCTCCTTGTGGTTGGA-3′; PKM1-sense, 5′-CGAGCCTCAAGTCACTCCAC-3′, and PKM1-antisense, 5′-GTGAGCAGACCTGCCAGACT-3′; PKM2-sense, 5′-ATTATTTGAGGAACTCCGCCGCCT-3′, and PKM2-antisense, 5′-ATTCCGGGTCACAGCAATGATGG-3′; GAPDH-sense, 5′-CCACCCATGGCAAATTCCATGGCA-3′, and GAPDH-antisense, 5′-TCTAGACGGCAGGTCAGGTCCACC-3′. GAPDH and RNU6B were used as internal controls. Normalization was not performed in the case of expression profiles of organ-specific miRNAs.

Total RNA was isolated from cultured cells or tumor tissues by using a NucleoSpin microRNA isolation kit (TaKaRa, Otsu, Japan) or Trizol reagent (Invitrogen) followed by DNase I treatment. RNA concentrations and purity were assessed by UV spectrophotometry. The primers for PTBP1, PKM1, PKM2, and GAPDH were the following:PTBP1-sense, 5′-ATC AGG CCT TCA TCG AGA TGC ACA-3′, and PTBP1-antisense, 5′-TGT CTT GAG CTC CTT GTG GTT GGA-3′; PKM1-sense, 5′-CGA GCC TCA AGT CAC TCC AC-3′, and PKM1-antisense, 5′-GTG AGC AGA CCT GCC AGA CT-3′; PKM2-sense, 5′-ATT ATT TGA GGA ACT CCG CCG CCT-3′, and PKM2-antisense, 5′-ATT CCG GGT CAC AGC AAT GAT GG-3′; GAPDH-sense, 5′-CCA CCC ATG GCA AAT TCC ATG GCA-3′, and GAPDH-antisense, 5′-TCT AGA CGG CAG GTC AGG TCC ACC-3′. RNU6B and GAPDH were used as an internal control. The relative expression levels were calculated by the ΔΔCt method.