TaqMan® MicroRNA Reverse Transcription Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Use 1 to 10 ng of total RNA per 15- µL RT reaction	Treat RT mixture for 30 minutes at 16C , 30 minutes at 42C and to inactivate treat the tube at 85C for 5 minutes

Upstream tips
Use 1 to 10 ng of total RNA per 15- µL RT reaction
Protocol tips
Treat RT mixture for 30 minutes at 16C , 30 minutes at 42C and to inactivate treat the tube at 85C for 5 minutes

Publication protocol

Total RNA from tissues or cultured cells were isolated using TRIzol reagent (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer's protocol. RNA concentration was measured using NanoDrop ND-1000 (Thermo Fisher Scientific) and the quality was assessed using electrophoresis with 1.5% denaturing agarose gels. TaqMan probe-based qPCR was performed using a commercial kit (Applied Biosystems, Thermo Fisher Scientific) according to the manufacturer's protocol. Reverse transcription was performed using a miR-125a-5p specific primer and ABI's TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Thermo Fisher Scientific). miR-125a-5p expression level was detected using a Taqman MicroRNA assay (Applied Biosystems, Thermo Fisher Scientific) with the Applied Biosystems 7900HT Sequence Detection system. U6 was used as the internal control. The RT–qPCR thermocycling conditions were as follows: 94°C for 30 s (initial denaturation), 94°C for 5 s (denaturation) and 55°C for 30 s (annealing), for 40 cycles. The following primers were used: miR-125a-5p forward, 5'-GGTAAGTCACGCGGT-3′ and reverse, 5'-CAGTGCGTCTCGTGGAGT-3′; U6 forward, 5'-CTGGTTAGTACTTGGACGGGAGAC-3′ and reverse, 5'-GTG CAGGGTCCGAGGT-3′. Subsequently, the 2−ΔΔCt method was used to quantify the expression level of the miRNA relative to U6.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for TaqMan® MicroRNA Reverse Transcription Kit below.

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