Reverace qpcr rt master mix with gdna remover

Four hepcidins identified from lined seahorse genome database (46 (link)) were cloned. All the reagents, consumables and anatomy tools were RNase free. Total RNA of seahorse liver was extracted following the instructions coming with the TRIzol Ragent (Invitrogen, Waltham, MA, USA). According to the operating steps of ReverAce qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan), 1 μg of total RNA was used to synthesize first-strand cDNA. All the total RNA and cDNA were stored at -80°C. The specific PCR primers of hampIIs were used to amplify and verify the coding sequences (Table S3). The parameters of specific polymerase chain reaction (PCR) were as follow: denaturation at 98°C for 5 min, followed by 36 cycles at 98°C for 30 min, 60°C for 30 s, and 72°C for 30 s, and finally extension for 10 min at 72°C. The DNA product were sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). Sequence alignments of HampII mature peptides were performed using MEGA.

Adult male non-pregnant (n = 4) and pregnant (n = 4) lined seahorses collected from a fish farm (Zhangzhou, Fujian, China) were anesthetized with MS222 before brood pouches sampling. After the separation of embryos, the brood pouch was washed with PBS to remove embryonic contamination and total RNA was extracted using TRIzol reagent according to the manufacturer’s instructions. 1 µg total RNA from the brood pouch sample was used to synthesize first-strand cDNA using the ReverAce qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). ‘No RT’ reactions were used as negative controls. The mRNA levels of genes involved in pregnancy, including C3.2, cd8a, cd79a, gata3, il12a, il12b, T-bet, and tgfb1, were determined by qRT-PCR (Supplementary Fig. 27). The primers used in this study are listed in Supplementary Table 18. qRT-PCR was performed on a Roche Light-Cycler 480 real time PCR system (Roche, Mannheim, Germany), using the SYBR Green I kit (Toyobo, Japan) according to the manufacturer’s instructions. β-actin was used as an internal control.