Mirna reaction buffer mix

miR-503 and U6 primers were purchased from Takara Bio (Otsu, Japan). Total miRNA was extracted from cultured cells and human tissue specimens with RNAiso for Small RNA (Takara Bio), according to the manufacturer’s instructions. PolyA tails were added to miR-503 and U6 with the miRNA Reaction Buffer Mix (Takara Bio) and cDNA was synthesised from 5 ng total RNA using the miRNA PrimeScriptRT Enzyme Mix (Takara Bio). Quantitative polymerase chain reaction (qPCR) was run on a CFX96™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with SYBR^® Premix Ex Taq™ II (Takara Bio). The qPCR conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec, then 60°C for 30 sec. The data were normalised against the U6snRNA. Subsequent to amplification, a melting curve analysis was performed to ensure the specificity of the products.

Total miRNA was extracted from cultured cells and human tissue specimens using RNAiso for Small RNA (TaKaRa Bio) according to the manufacturer's instructions. PolyA tails were added to miR-34a and U6 using the miRNA Reaction Buffer Mix (TaKaRa Bio), and cDNA was synthesized from 5 ng of total RNA using the miRNA Prime ScriptRT Enzyme Mix (TaKaRaBio). Real-time PCR was performed on a CFX96™ Real-Time PCR Detection System (Bio-Rad) using SYBR® Premix Ex Taq™ II (TaKaRa Bio). The following PCR conditions were used: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The data were normalized against the expression of the U6snRNA. After amplification, melting curve analysis was performed to ensure the specificity of the products.

The primers for miR-338-3p and U6 were produced using the miScript Primer Assay kit (Qiagen Dusseldorf Germany). The sequences of the miRNAs used in this study were as follows: miR-338-3p: UCCAGCAUC- AGUGAUUUUGUUG and U6: CGCAAGGAUGACACGCAAAUUCGUGAA- GCGUUCCAUAUUUUU. The reverse primers were also used in the reverse transcription step. Total miRNA was extracted from cultured cells and human tissue specimens using RNAiso for Small RNA (TaKaRa Bio,Otsu, Japan) according to the manufacturer's instructions. Poly-A tails were added to miR-338 and U6 with the miRNA Reaction Buffer Mix (TaKaRa Bio), and then cDNA was synthesized from 5 ng of total RNA using the miRNA PrimeScript RT Enzyme Mix (TaKaRa Bio). Real-time PCR was performed in a CFX96 Real-Time PCR Detection System (Bio-Rad) with SYBR Premix Ex Taq II (TaKaRa Bio). The PCR conditions were 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The data were normalized against the U6 snRNA. After amplification, a melting curve analysis was performed to confirm the specificity of the products.