Taqman mir assays for cel mir 54

Relative quantification of miR-122 was carried out using the qRT-PCR miR Detection Kit (Ambion®, Life Technologies, USA) and hsa-miR-122 PCR Primer Sets for amplification of the miR-122. qRT-PCR was performed using the Applied Biosystems Step One Plus Real-Time PCR System taking advantage of the Taqman miR Assays for cel-miR-54, miR-122, and the Taqman Universal Master Mix II no UNG (all Applied Biosystems, Carlsbad, USA) in a final volume of 20 µl including 1 µl cDNA from the RT reaction as template. Relative expression of miR-122 with cel-miR-54 as control was expressed using the comparative 2^-ΔCT method. PCR conditions were as follows: incubation of the samples for 10 min at 95°C followed by application of 40 cycles of 15 seconds at 95°C and 1 minute at 60°C and all samples were run in duplicate.

Relative quantification of miR-122 was carried out using the a qRT-PCR miR Detection Kit (Ambion®, Life Technologies, USA) using hsa-miR-122 PCR Primer Sets for amplification of the miR-122. qPCR was performed using the Applied Biosystems Step One Plus Real-Time PCR System taking advantage of the Taqman miR Assays for cel-miR-54, miR-122, and the Taqman Universal Master Mix II no UNG (all Applied Biosystems, Carlsbad, USA) in a final volume of 20 μl including 1 μl cDNA from the RT reaction as template. All samples were run in duplicate. PCR conditions were as follows: incubation of the samples for 10 min at 95 °C followed by application of 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Relative expression of miR-122 with cel-miR-54 as control was expressed using the comparative CT method using 2^-ΔCT [28 (link)]. Moreover, miRNA quality was not influenced by duration of storage since all samples were analysed in duplicate and no relevant deviation was detectable (p = 0.897). Finally, regression analysis with storage duration and miR-122 expression revealed no significant association of storage duration and miRNA-122 expression (p = 0.526).