Trizol total rna extraction reagent

In order to obtain RNA samples for further experiments, a total of six different tissues (corpus luteum, large follicle, skeletal muscle, uterus, ovary, and mammary gland) of the goat were collected (n = 3). Total RNA extraction was carried out using TRIzol total RNA extraction reagent (Takara, Dalian, China), following the instructions of the manufacturer. Then, 1% agarose gel electrophoresis in 6 × loading buffer was used to evaluate the integrity of total RNA. RNA was then quantified and qualitatively analyzed using a Nanodrop 2000 Spectrophotometer. Qualified RNA samples were stored at −80°C. To accomplish the further experiment, first-strand cDNA was synthesized by Prime Script™ RT Reagent Kit (Takara, Dalian, China). After that, these cDNAs were conserved at −20°C for subsequent experiments.
Shaanbei white cashmere (SBWC) female goats (2–3 years) that were healthy and under the same feeding and management conditions were used as the DNA sample in this work. All genomic DNAs were isolated from the ear tissue of 312 female goats according to the protocol of our previous study (29 (link)) and then stored at −40°C. In addition, all litter size and growth trait records of these goats were provided by the staff from farm records.

Eruca seeds of PI 426649 and PI 426652 originally from the Agricultural Research Service, USDA were germinated on filter paper immersed in liquid MS medium [87 (link)] without sugar or organic components. Seven days later the seedlings were treated for 10 h with 20% PEG-6000/liquid MS and then harvested and frozen imediately in liquid nitrogen and then stored at − 80 °C. Drought-tolerant PI 426649 is denoted as ‘DT and Drought-sensitive PI 426652 is denoted as ‘DS’. Treatment with liquid MS medium is denoted as ‘MS’ while treatment with PEG is denoted as ‘PEG’. Four samples (DT-MS, DT-PEG, DS-MS, DS-PEG) each with two biological replicates were taken for the present study. Total RNA was isolated from the whole seedlings that had been stored at − 80 °C by using TRIZOL total RNA extraction reagent (TAKARA) according to the manufacturer’s protocol. RNA integrity was verified by 1.5% Agrose gel electrophoresis and confirmed using a 2100 Bioanalyzer analyzer (Agilent, CA, USA). The mRNA enrichment, RNA fragmentation, the first and second strand cDNA synthesis and purfying, sequencing adaptors ligation and PCR amplification were performed as described [14 (link)]. The libraries were applied to Illumina sequencing platform (HiSeq 2000, SanDiego, CA, USA) for high-throughput sequencing using a paired-end read protocol with 100 bp of data collected per run.

Total RNA was extracted from the fresh Fallopian tube samples using Trizol total RNA extraction reagent (Takara, Dalian, China). Agarose gel electrophoresis was used to ascertain the quality of the RNA products by the presence of 28S rRNA, 18S rRNA and 5S rRNA bands, with a 2:1 ratio of 28S rRNA to 18S rRNA. cDNA was reverse transcribed from the extracted RNA using RT kit (Takara, Dalian, China). The primers used for EphA2 were 5’-AAGACCCTGGCTGACTTT-3′ (forward) and 5’-GTTCACCTGGTCCTTGAGT-3′ (reverse), while the primers for 18sRNA were 5’-CCTGGATACCGCAGCTAGGA-3′ (forward) and 5’-CCTGGATACCGCAGCTAGGA-3′ (reverse). Real time RT-PCR was carried out using an ABI 7300 real-time PCR system (ABI, Carlsbad, USA) with 5 μl diluted cDNA and 0.5 μl primers. The programmed thermocycler included 40 cycles of predenaturation at 95 °C for 10 min, followed by denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, and extension at 72 °C for 30 s. All reactions were performed in triplicate. Relative quantification of EphA2 mRNA was calculated using the comparative Ct method (ΔΔCt = ΔCt _EphA2− ΔCt_18sRNA).