Quantitative Analysis of JH Key Genes

To analyze the effect of JH on the JH signal key gene in soldiers, Quantitative Reverse Transcription PCR was performed using the sample of the soldiers’ head on the 1st to 4th day after feeding JHA. Met, Kr-h1 and RaSsp1 were selected as JH key genes. The detail of qRT-PCR was the same as in a previous report [17 (link)]. RNA was extracted from 5 heads of soldiers with Trizol RNA extraction reagent (Thermo, Carlsbad, CA, USA), and then cDNA was obtained using the reverse transcription kit HiScript III RT SuperMix for qRT-PCR (Vazyme Bio, Nanjing, China). qRT-PCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR system (ABI, Foster City, CA, USA). Ribosomal protein gene L13a (RPL13a) and elongation factor 1α (EF1-α) mRNA levels were used as references. The specific primers were as follows, RaSsp1: F 5′-ACTGTGCTTGGCGCTGTC-3′, R 5′-CTGGGATGTGGTATTGCTT T-3′; Met: F 5′-GCCCTCATCATCCGCCTT, R 5′-CTTGCCATCACGAGAACG-3′; Kr-h1: F 5′-AGCAGCCCAGATTTACCT, R 5′-GTCTTCGCCCTCCTTTCC-3′; RPL13a: F 5′-TCTGTGGAGGACGGTTAG, R 5′-ACTTTCTGCCTGGTTTCA-3′; and EF1-alfa: F 5′-CCCTTCGTCTTCCTCTTC, R 5′-CTCCAGCGACATAACCAG-3′. Each experiment was performed with three biological replicates, and three qRT-PCR analyses were performed using the same cDNA sample of each time point.