Total RNA from cultured cells or tissues was isolated using the Qiagen miRNeasy kit. RNA was reverse transcribed with Moloney-murine leukemia virus (Promega). Real-time PCR for miRNA and mRNA were performed as described before (17 (link)). Briefly, mRNA SYBR green quantitative real-time PCR (Applied Biosystems) was performed to detect expression of mRNA levels in a HT7900 Fast Real-Time PCR System. 18S was used as the internal control. miRNA quantitative real-time PCR was performed according to the instruction of miRNA assay kit (Applied Biosystems) with snoRNA202 as the internal control.
Protocol detail
Quantitative Real-Time PCR for miRNA and mRNA
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Assay
Cultured cells
Mirna
Moloney murine leukemia virus
Mrna
Promega
Quantitative real time pcr
Sybr green
Tissues
Corresponding Organization : Institute of Molecular and Cell Biology
Other organizations : Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Brigham and Women's Hospital, University of Geneva
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Variable analysis
independent variables
- None explicitly mentioned
- MRNA levels
- MiRNA levels
- 18S as internal control for mRNA quantification
- SnoRNA202 as internal control for miRNA quantification
- Positive control: None mentioned
- Negative control: None mentioned
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