The following miRCURY® LNA® miRNA PCR Assays (QIAGEN, Milan, Italy; Cat. no. 339306) were selected: gga-miR-18a-5p (assay ID-YP02100185); gga-miR-18b-5p (assay ID-YP02100265); hsa-miR-22-3p (assay ID–YP00204606); cbr-miR-124 (assay ID–YP02103368); has-miR-145-5p (assay ID–YP00204483); has-miR-21-5p (assay ID–YP00204230); has-miR-146b-5p (assay ID–YP02119310). Assays used as reference controls for miRNA normalization included: hsa-miR-16-5p (assay ID–YP00205702) and hsa-let-7a-5p (assay ID–YP00205727) [29 (link),36 (link)].
RT-qPCR was performed using miRCURY LNA SYBR Green PCR Kit (QIAGEN, cat. no. 339345, 339346) following the manufacturer’s instructions. Quantitative reaction was performed in duplicate per each sample in a 20 μL total reaction volume, containing 10 μL of 2× miRCURY SYBR Green Master Mix, 2 μL of specific miRNA assay, 2 μL of RNase-free water and 6 μL of cDNA (60× diluted), and performed using a Rotor-Gene® Real-Time PCR system (QIAGEN). The cycling conditions were set as follows: 95 °C for 2 min, 40 cycles of 95 °C for 10 s and 56 °C for 60 s. MiRNA expression levels were presented in terms of fold change normalized to endogenous controls using the formula .
RT-qPCR was performed using miRCURY LNA SYBR Green PCR Kit (QIAGEN, cat. no. 339345, 339346) following the manufacturer’s instructions. Quantitative reaction was performed in duplicate per each sample in a 20 μL total reaction volume, containing 10 μL of 2× miRCURY SYBR Green Master Mix, 2 μL of specific miRNA assay, 2 μL of RNase-free water and 6 μL of cDNA (60× diluted), and performed using a Rotor-Gene® Real-Time PCR system (QIAGEN). The cycling conditions were set as follows: 95 °C for 2 min, 40 cycles of 95 °C for 10 s and 56 °C for 60 s. MiRNA expression levels were presented in terms of fold change normalized to endogenous controls using the formula .
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