Total RNA, including small RNA, was extracted and purified using the miRNeasy Mini Kit (QIAGEN Inc., Valencia, CA, USA) following the manufacturer's instructions. For miRNA analysis, TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) was first used to generate cDNA with the hairpin primers, which are specific to the mature miRNA and will not bind to the precursors. The expression levels of miR-125a-5p, miR-125b, and miR-205 were then measured by real-time PCR using TaqMan MicroRNA Assays (assay ID: 002198, 000449, 000509, respectively; Applied Biosystems) according to the manufacturer's protocol. RNU6B was used as an internal control to normalize all data using the TaqMan RNU6B Assay (assay ID: 001093; Applied Biosystems). RNU6B levels were unaffected by entinostat treatment. The relative miRNA levels were calculated using the comparative Ct method (ΔΔCt).
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miRNA Expression Analysis via RT-PCR
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Corresponding Organization :
Other organizations : University of Colorado Anschutz Medical Campus, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Memorial Hospital of South Bend, Fuzhou General Hospital of Nanjing Military Command
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Variable analysis
independent variables
- Entinostat treatment
- Expression levels of miR-125a-5p
- Expression levels of miR-125b
- Expression levels of miR-205
- RNU6B levels
- RNU6B was used as an internal control to normalize all data using the TaqMan RNU6B Assay (assay ID: 001093; Applied Biosystems)
- RNU6B levels were unaffected by entinostat treatment
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