Oligotex mrna kit

mRNA was isolated from Z1-M using an Oligotex mRNA Kit (Qiagen, Hilden, Germany) and full-length cDNA was synthesized by cDNA Library Construction Kit (Takara, Kyoto, Japan). A pair of specific primers (Supplementary Material 1) was designed for each new transcript using the Primer Premier 5 software, and then sent to Sangon Biotech (Shanghai, China) for synthesis. The synthesized full-length cDNA was used as a template for PCR. The sequences could be amplified with the target bands as true transcripts.
Three female and three male zebrafish were used to construct three full-sib groups in a one to one way. 30 individual samples were harvested at unfertilized egg, 2-cell, high, 80%-epiboly, 3-somite, prim-5, and protruding-mouth stages. Samples at the same stage from 3 full-sib groups were mixed together, with seven samples ultimately obtained. For these samples, we used the same methods described above to extract the total RNA and to synthesize cDNA. Using these cDNAs as templates, RT-qPCR was used to detect changes in the expression levels of new transcripts that significantly decreased at the prim-5 stage compared to the unfertilized egg stage. RT-qPCR data were analyzed by two-tailed independent t-test. And P < 0.05 was considered to be statistically significant.

Expression experiments were performed in both male and male tissues for the Y-linked genes that lack high similarity to their female homologs. Three male and three female grass carp individuals (18 months old) were selected from the full-sib population. Brain, hypothalamus, gonad, and pituitary samples were prepared, equal quantities of the three samples from the same tissue were mixed, resulting in eight samples. RNA was isolated with Oligotex mRNA Kit (QIAGEN, Germany) in accordance with the manufacturer’s protocol. Primers were designed using Primer5 and synthesized by Sangon Company (Shanghai, China). The primers are listed in Supplementary Table S7. The PCR reaction system was 20 μL in volume, and the PCR program was as follows: initial denaturation at 94 °C for 2 min, denaturation at 94 °C for 30 s, annealing at 51 °C for 30 s, and extension at 72 °C for 30 s. After 39 cycles, the program was extended at 72 °C for 2 min. Protein sequences were aligned using ClustalW2⁴⁵ , and a Maximum Likelihood tree with Poisson correction was constructed using MEGA6^{46 (link)}.

Two different approaches have been tested. The data presented in this manuscript has been generated from samples using the following strategy: The Oligotex mRNA kit (Qiagen, Hilden, Germany) was used to isolate the algal mRNA. For isolation of D. shibae mRNA, the MICROBEnrich kit (Ambion, Life Technologies, Darmstadt, Germany) was used to remove the eukaryotic rRNA, and the MICROBExpress kit (Ambion, Life Technologies, Darmstadt, Germany) was used to remove the prokaryotic rRNA, according to the manufacturer's instructions. A second approach resulted in an mRNA proportion of about 0.01%. These samples have therefore not been analyzed further: PolyATract System IV (Promega, Madsion, WI, USA) was used for isolation of algal mRNA according to the manufacturer's instructions. For isolation of the bacterial mRNA Ribo-Zero Magnetic Kit for both plant leaf and gram-negative bacteria (Epicenter, Madsion, WI, USA) were used according to the manufacturer's instructions. The final purification of the eluted mRNA was performed using ethanol precipitation.