Trizol lysis

Gene expression levels of IgM and IgT after immunization were conducted. Total RNA extracted from each tissue (gill, gut, and kidney) was prepared by TRIzol lysis (Invitrogen, USA). cDNA was then synthesized according to the protocol of Reverse Transcriptase M-MLV Kit (TaKaRa, Dalian, China). The RT-PCR was performed using THUNDERBIRD SYBR qPCR Mix Kit (TOYOBO, Shanghai, China). The primer was designed following previous reports (15 (link), 16 (link)).

Prior to RNA extraction, pituitary tissue samples were placed into bead mill tubes containing six 1.4-mm ceramic beads (MoBio Laboratories) and homogenized for 30 s at 6.5 m/s at 4 °C using an Omni Bead Ruptor 24 bead mill. RNA was extracted with the NucleoSpin® miRNA kit (Macherey Nagel) in combination with TRIzol lysis (Invitrogen) following the manufacturers’ protocols, allowing for collection of small RNA (< 200 nt) and large RNA (> 200 nt) simultaneously into separate tubes from total RNA. RNA quantity was determined using Nanodrop and the quality was assessed by an Agilent 2100 Bioanalyzer.

In this study, we used the following TaqMan probes (Thermo Fisher Scientific): GAPDH (Hs03929097_g1), DR4 (Hs00234355_m1), TNR1 (Hs00533568_g1), FAS (Hs00236330_m1), and BID (Hs00609632_m1).
After 18 hours of perifosine treatment in vitro , the cells were subjected to Trizol lysis (Thermo Fisher Scientific; CAT 15596018) following the manufacturer’s specifications. cDNA synthesis was performed using Super Script III kit reagents ( Thermo Fisher Scientific; CAT 18080051), and qPCR reactions were performed using TaqMan Universal Master Mix II with UNG kits (Thermo Fisher Scientific; CAT 4440038), according to the manufacturer’s instructions. GAPDH was used as the housekeeping gene, and gene expression was calculated using the formula 2 ^-ΔΔCT . The reactions were performed on an ABI Prism 7500 thermocycler (Thermo Fisher Scientific).

Cell sediments were collected, and RNA sediments were extracted using Trizol lysis (Thermo, USA). RNA samples were reverse transcribed according to the Reverse Transcription Kit (Yeasen, Shanghai, China). The reverse transcribed cDNA samples were diluted 5-10-fold with DEPC and stored at -20 °C. Next, real-time fluorescence quantitative PCR was performed using SYBR Green Master Mix (Yeasen, Shanghai, China) and determined on an ABI Prism 7500 Sequence Detection System (Applied Biosystems, USA). Quantitative expression levels were analyzed using the 2^−ΔΔCt method and normalized with β-actin. Primer sequences are shown in Table 1 of supplementary materials.