4.5. Design and Synthesis of Primers and Gene Sequencing

HIF-1α is respond to trigger and coordinate multiple gene regulation under low oxygen or low temperature signals. To identify the target gene HIF-1α, the PCR amplification and sequencing were carried out as the following steps. After total RNA was treated with DNase I (Promega, Madison, USA), cDNA was synthesized by reverse transcription according to the instructions of PrimeScript^TM 1st Strand cDNA Synthesis Kit (Takara, Takara Biomedical Technology (Dalian) Co., Ltd., Dalian city, China) and stored at −20 °C. Primers for the target gene HIF-1α were designed using the online software OligoArchitectTM Online (http://www.oligoarchitect.com/, accessed on April 2020) that resulted in forward primer 5′-GGCGATACAGATAACAACAA-3’(1α-F) and reverse primer 5′-TCTCCTTCTCCTTCACTTG-3′ (1α-R). The amplified fragment size was of 96 bps for the target gene.

18S rRNA was used as an internal reference gene in RT-PCR [31]. The sequences of the forward and reverse primers were 5′-ATCACGGTGCTCTTTACT-3′ (18S-F) and 5′-CGAGATCCTATTCCATTATTCC-3′ (18S-R), respectively. The amplified product fragment was of 124 bps by the two 18S rRNA primers. Both primer synthesis and gene sequencing measurement were performed by Wuhan Qingke Innovation Biotechnology Co., Ltd.