Total RNA was isolated from the frozen tissue samples and the cultured cells using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. Then, cDNA was synthesized from 100 ng of the RNA, using a BcaBEST RNA PCR kit (Takara, Dalian, People’s Republic of China) according to the manufacturer’s instruction. The cDNAs were subjected to the quantitative reverse transcription polymerase chain reaction (qRT-PCR) using SYBR Premix Ex Taq (Takara) to detect miR-613 and SOX9 mRNA with the ABI 7900 Fast system (Thermo Fisher Scientific). The primers used in this study have been described previously.15 (link),22 (link) The U6 gene was used as an endogenous control for miRNA expression,25 (link) whereas the GAPDH gene was used as an endogenous control for mRNA expression. The relative expression levels of miR-613 and SOX9 mRNA were calculated using the comparative delta CT (2−ΔΔCT) method.26 (link)
Protocol detail
Quantitative Analysis of miR-613 and SOX9
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Cdnas
Cultured cells
Frozen
Gapdh
Gene
Mirna
Mrna
Primers
Reverse transcription polymerase chain reaction
Sox9
Tissue
Trizol
Corresponding Organization :
Other organizations : Jilin University, First Hospital of Jilin University
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Variable analysis
independent variables
- None explicitly mentioned
- Expression levels of miR-613
- Expression levels of SOX9 mRNA
- Expression levels of U6 gene (for miRNA expression)
- Expression levels of GAPDH gene (for mRNA expression)
- Positive control: Not mentioned
- Negative control: Not mentioned
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