Aliquots of 10 ng of eluted RNA samples were used to make cDNA using the TaqManTM MicroRNA Reverse Transcription Kit (Applied Biosystems, USA) and 5X RT Human TaqMan MicroRNA Assay primers (hsa-miR-21-5p: Assay ID_000397; RNU6B: Assay ID_001093) according to the manufacturer’s instructions. The reaction was performed in a Veriti thermal cycler (Applied Biosystems, USA) for 30 minutes at 16°C followed by 30 minutes at 42°C and an additional 5 minutes at 85°C.
Quantitative PCR was performed on a 7500 Real Time PCR system (Applied Biosystems, USA) with TaqMan™ Universal PCR Master Mix (Applied Biosystems, USA), 20X primers and nuclease-free water to a total volume of 10μL. The cycling process was performed as follows: 50°C for 2 minutes and incubation at 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. The threshold standard deviation (SD) for intra-assay and interassay replicates was 0.3.
All samples were analyzed in triplicate on the ABI 7500 platform (Applied Biosystems, USA). The cycle quantification (Cq) values were calculated with SDS 1.4 software (Applied Biosystems).
Quantitative PCR was performed on a 7500 Real Time PCR system (Applied Biosystems, USA) with TaqMan™ Universal PCR Master Mix (Applied Biosystems, USA), 20X primers and nuclease-free water to a total volume of 10μL. The cycling process was performed as follows: 50°C for 2 minutes and incubation at 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. The threshold standard deviation (SD) for intra-assay and interassay replicates was 0.3.
All samples were analyzed in triplicate on the ABI 7500 platform (Applied Biosystems, USA). The cycle quantification (Cq) values were calculated with SDS 1.4 software (Applied Biosystems).