2.5. Validation and quantification of miR expression by miR TaqMan reverse transcription PCR

Real‐time reverse transcription PCR (RT‐PCR) was performed to validate differentially expressed miRs in osteosarcoma cell lines. Expressions of miR‐15b were determined in osteosarcoma tissues. For mature miR detection, cDNA reverse transcription was performed from total RNA samples using specific miR primers from the TaqMan miR Assay and reagents from the TaqMan miR Reverse Transcription kit (Life Technologies, Foster City, CA, USA). The resulting cDNA was amplified by PCR using TaqMan miR Assay primers with the TaqMan Universal PCR Master Mix and analysed with a StepOnePlus Real‐Time PCR System (Life Technologies) according to the manufacturer's instructions. Expression of RNU48 miR was used as an endogenous control. The relative levels of miR‐15b expression were calculated from the relevant signals by normalization with the signal for RNU48 miR expression. All reactions were performed in triplicate. The relationships between miR‐15b expression levels and clinical features of patients with osteosarcoma were analysed by chi‐square test and Student's t‐test. The miR‐15b expression levels were divided by the median value. The high‐expression group (> 600) was set above the median value, and the low‐expression group (≤ 600) was set below the median value. Overall survival curves were analysed with the Kaplan–Meier method and compared using the log‐rank test. A statistically significant difference was defined as P‐value less than 0.05.