qRT-PCR
Description
To assess whether the differentially expressed miRNAs were consistent with the gene chip results, we validated rno-miR-3068-3p expression via qRT‒PCR. The results revealed that the rno-miR-3068-3p expression level was higher in the I/R group than in the sham group and that CIP led to the downregulation of rno-miR-3068-3p expression in rat hippocampal tissues (P < 0.05) . The trend of the rno-miR-3068-3p change was consistent with the gene chip results, confirming the reliability of the gene chip study results.
Files
Steps to reproduce
Eight hippocampal tissue samples were randomly selected from each group. Total RNA was extracted using the TRIzol method, and RNA integrity was determined via agarose gel electrophoresis. The concentration and purity of the RNA were assessed using a UV‒visible spectrophotometer. Reverse transcription reactions were performed using a reverse transcription kit (TaKaRa, Dalian) according to the manufacturer’s instructions. qRT-PCR was performed using SYBR® Premix Ex Taq™ II kit (TaKaRa, Dalian). Each reaction was performed in a 20 µl system containing 2.0 µl of cDNA, 0.8 µl of forward and reverse primers, 10 µl of SYBR® Premix Ex Taq II, 0.4 µl of ROX Reference Dye and 6 µl of RNase-free water. The PCR conditions were as follows: denaturation at 95°C for 30 s followed by 40 cycles of denaturation at 95°C for 5 s and extension at 60°C for 30 s. Human U6 small nucleolar RNA was used as the internal reference gene.