Taqman microrna reverse transcription kit

Total RNA was extracted from tissues using the TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's instructions. RNA concentration and purity was assessed using a NanoDrop ND-1000 (Thermo Fisher Scientific, Inc.). A total of10 ng total RNA was used for cDNA synthesis using the Taqman MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). Reverse transcription with a miR-124a-specific primer was performed using ABI's Taqman MicroRNA Reverse Transcription kit, miR-124a expression level was detected using a Taqman MicroRNA assay (Applied Biosystems; Thermo Fisher Scientific, Inc.) (19 (link)). The RT-qPCR thermocycling conditions were as follows: 94°C for 30 sec (initial denaturation), 94°C for 5 sec (denaturation) and 55°C for 30 sec (annealing), for 40 cycles. U6 expression was used as the internal control. The following primers were used: miR-124a forward, 5′-GGTAAGGCACGCGGT-3′, and reverse, 5′-CAGTGCGTGTCGTGGAGT-3′; U6 forward, 5′-CTGGTTAGTACTTGGACGGGAGAC-3′, and reverse, 5′-GTGCAGGGTCCGAGGT-3′. All reactions were performed three times in triplicate, and relative expression of miRNAs was calculated using the standard curve and the ΔΔCq method (20 (link)).

To detectCASC11 and miRNA-182, total RNA and miRNA extractions were performed using RNAzol^® RT RNA Isolation Reagent (Sigma-Aldrich‎) and mirVana miRNA Isolation Kit (Thermo Fisher Scientific), respectively. Reverse transcriptions were performed using AMV Reverse Transcriptase (NEB, USA) and TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific), respectively. In total RNA and miRNA reverse transcriptions, 1.2ug total RNAs and 100 ng miRNAs were used in 20 ul system. Luna^® Universal One-Step RT-qPCR Kit (NEB) and TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific) were used to prepare PCR mixtures. 1 ul cDNA was added into 20 ul PCR reaction system. Expression of CASC11 and miRNA-182 was normalized using 2^−ΔΔCT method.

Total miRNA was extracted from liver cancer cells using the QIAGEN miRNeasy kit (Qiagen K.K.) for miRNA quantification according to the instructions provided by the manufacturer. Total RNA was reverse-transcribed using TaqMan MicroRNA Reverse Transcription kit (Life Technologies Japan). RNU6B was used as an internal control for relative quantification of hsa-miR-30d.
Circulating miRNA was extracted from 200 µl of serum samples using the Qiagen miRNeasy serum-plasma kit (Qiagen K.K.) according to the instructions provided by the manufacturer. RNA was reverse-transcribed using the TaqMan MicroRNA Reverse Transcription kit (Life Technologies Japan) following the manufacturer's instructions. Caenorhabditis elegans miR-39 (cel-miR-39) was spiked in each sample as a control for the extraction and amplification steps. Serum miRNA was amplified using primers and probes provided by Applied Biosystems by the TaqMan MicroRNA assay, according to the instructions provided by the manufacturer. The relative expression of serum miRNA was calculated using the comparative cycle quantification (Cq) method (2^−ΔΔCq) (18 (link)) with spiked cel-miR-39 as a normalized internal control.