RNA was extracted as described previously (10 (link)) and in SI Appendix . Two hundred fifty nanogram RNA was reversed transcribed using stem-loop Multiplex primer pools (Applied Biosystems). Reverse-transcriptase-specific primers for mmu-miR-335-5p (Applied Biosystems miRNA assay ID 000546), mmu-miR-134-5p (ID 001186), mmu-miR-22-3p (ID 000398), mmu-miR-132-3p (ID 000457), and mmu-miR146a-5p (ID 000468) were used and real-time quantitative PCR performed using TaqMan miRNA assays (Applied Biosystems) on the QuantStudio™ Flex PCR system (Thermo Fisher Scientific). Comparative CT values were measured. MiRNA levels were normalized using U6B (Applied Biosystems miRNA assay ID 001093) or RNU19 (Applied Biosystems miRNA assay ID 001003) expression, and relative fold change in miRNA levels were calculated using the comparative cycle threshold method (2−ΔΔCT). Ago2 immunoprecipitation and small RNA sequencing methods are as described previously (10 (link)) and are detailed in SI Appendix .
Protocol detail
Quantification of miRNA Expression
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Ago2
Assay
Immunoprecipitation
Mirna
Mmu mir 132
Mmu mir 134
Mmu mir 22
Mmu mir 335
Primers
Real time pcr
Reverse transcriptase
Stem
Corresponding Organization :
Other organizations : Royal College of Surgeons in Ireland, Science Foundation Ireland, Aarhus University, Omiics (Denmark), Philipps University of Marburg, University Hospital Frankfurt, Goethe University Frankfurt, University of Rostock, University of Southern Denmark, Odense University Hospital, Queen's University Belfast, University College London
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Variable analysis
independent variables
- None explicitly mentioned
- Levels of mmu-miR-335-5p, mmu-miR-134-5p, mmu-miR-22-3p, mmu-miR-132-3p, and mmu-miR146a-5p
- U6B expression
- RNU19 expression
- Positive control: None mentioned
- Negative control: None mentioned
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