Dnase 1 treatment

Total RNA was prepared from 40 mL exponentially growing cell cultures using the UNIQ-10 Trizol RNA extraction kit (Sangon Biological Engineering, Shanghai, China). The samples were digested using DNase I treatment (TakaRa, Dalian, China) to avoid DNA contamination. The treated total RNA was used for cDNA synthesis using the PrimeScript RT-PCR Kit (TakaRa, Dalian, China). qPCR was performed using the SYBR Green Master Mix Kit (Roche Molecular Biochemicals, Germany). The ACT1 gene was chosen as the internal control gene. The relative transcription levels of genes were analyzed using the 2^−ΔΔCT method.

For quantitative fluorogenic RT-PCR (qRT-PCR), total RNA was isolated with RNeasy Mini kits with an on-column RNase-free DNase I treatment (Takara). Total RNA was reverse transcribed with random hexamers and oligo (dT) primers. Diluted reactions were analyzed with SYBR green PCR reagents (Takara, Dalian, China). Endogenous mouse EphB2 and exogenous EphB2-Flag mRNA empty were normalized to GAPDH. The following primers were used: mouse EphB2 forward, 5′-GTGTGGAGCTATGGCATCGT-3′ reverse, 5′-TGGGCGGAGGTAGTCTGTAG-3′ Flag; forward, 5′-ATTCTGCTGGCTGCTGCT-3′ reverse, 5′-CGTTGCTGTCGTAGAGTCC-3′.

The methodology for total RNA extraction and qRT-PCR was conducted according to the procedures described in a previous study [24 (link)]. To ensure the absence of genomic DNA contamination, the isolated RNA was subjected to DNase I treatment (Takara, Beijing, China). First-strand cDNAs were synthesized using a reverse transcription PCR cDNA synthesis kit (Takara, China). Triplicate independent qRT-PCR analyses were performed using the Roche Lightcycler 96 Fluorescence Quantitative PCR Instrument and TB Green Premix Ex TaqTM II (Takara, China). The ACT1 gene served as the internal control.

Total RNA was extracted from the SW480
cell line using Trizol according to the manufacturer’s
protocol. The RNA quality and yield
of extraction was analyzed by agarose gel electrophoresis
(BioER, China) and spectrophotometry
(Gene Quest, England) respectively.
In order to remove any genomic traces, DNaseI
treatment (Takara, Japan) was performed prior
to cDNA synthesis at 37˚C for 30 minutes followed by heat and Ethylenediamine tetra acetic
acid (EDTA, Sigma, USA) inactivation. cDNA
was then synthesized by PrimeScript II reverse
transcriptase (Takara, Japan) according to the
manufacturer’s protocol. For miRNA detection,
polyA tail was added to the 3´ end of RNAs before
cDNA synthesis by using polyA polymerase
(Takara, Japan) according to the manufacturer’s
protocol. The anchored oligodT (5´-GCGTCGACTAGTACAACTCAAGGTTCTTCCAGTCACGACGTTTTTTTTTTTTTTTTTT-
3´) was
used for cDNA synthesis. Real-time quantitative
PCR was performed using standard protocols
on an ABI PRISM 7500 instrument (Applied
Biosystems, USA). The following primers
were used for real-time PCR: miR-590-5p
forward 5´-GAGCTTATTCATAAAAGTG-3´, U48 forward 5´-TGACCCCAGGTAACTCTGAGTGTGT-3´ and the Universal primer: 5´-AACTCAAGGTTCTTCCAGTCACG-3´. Output data were analyzed by GraphPad Prism. U48 SNO-RNA expression levels were used as internal control for normalization of miR-590 expression.

Total RNA was isolated from 9 brain tissues using TRIzol® Reagent following the manufacturer’s instructions (Invitrogen, CA, USA), and DNase I treatment (TaKara, Tokyo, Japan) was employed. After ensuring quality and quantification, high-quality RNA samples (OD260/280 = 1.8 ∼ 2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28 S:18 S ≥ 1.0, > 10 µg) were used for constructing the sequencing libraries.

The total RNA were extracted from fish liver and intestine samples using the TRIzol reagent (Invitrogen, USA). The concentration and integrity of these RNA samples were detected through spectrophotometry and electrophoresis following the procedures outlined by Wu et al. (32 (link)). After treatment with DNase I treatment (Takara, Dalian, China), 10 µg of total RNA samples were reverse transcribed with SuperScript™ II RT (Takara, Dalian, China) in 75 µL reactions. Primers for quantitative real-time PCR were designed according to largemouth bass genes (Table 2). Primer synthesis was carried out by Biosune Co. (Shanghai, China). β-actin was served as the internal reference. Quantitative PCR (qPCR) assays were conducted following the methods described by Wu et al. (16 (link)) and Yang et al. (33 (link)). Each analysis was performed carried out in at least triplicate.