Abi 7500

MDA-9 shRNA and control shRNA were purchased from Open Biosystems. HEK293T cells were seeded in a 6-well dish and then transfected with 1μg of retroviral DNA encoding MDA-9 vector or control vector, 1 μg packaging plasmid mix (0.8 μg psRAX2 and 0.2 μg pMD2.G) to generate lentiviruses. Virus infection was performed in MDA-MB-231 cells for 48 h. Add puromycin to screen for virus-infected cells. Cells were then collected and total RNA was extracted. RNA extraction was performed using SV Total RNA (Promega, Beijing, China). cDNA was reserved from total RNA by using the First Strand cDNA Synthesis Kit (Thermo, Shanghai, China). The Real-time PCR (Applied Biosystems ABI 7500, Waltham, MA, USA) and SYBR-Green PCR Master Mix (Takara, Beijing, China) were used for amplification and detection. GAPDH was used as the normalization control of the amplifications.

About 5 × 10⁸ cells in each of four groups were collected, 500 μl TRIzol (R0016, Beyotime) lysate protein nucleic acid complex was added, and 100 μl chloroform was mixed to extract RNA. After centrifugation at 12000 RPM, 4°C, for 10 min, the upper colorless aqueous phase was collected, 200 μl isopropanol was added to precipitate RNA, and 75% of the cells were washed. The extracted RNA was retrotranscribed into a cDNA template with a reverse transcription reagent (RR037Q, Takara). Then, a real-time fluorescent quantitative polymerase chain reaction (Applied Biosystems, ABI 7500) was conducted with TB Green qPCR reagent (RR82LR, Takara). The Ct value of genes to be examined was calibrated using the Ct value of the internal reference gene GAPDH, and the amplification multiple of genes relative to the internal reference genes was calculated according to the 2^−∆∆Ct formula. Amplification conditions: the first stage was kept at 95°C for 30 s; the second stage was 95°C for 5 s and 60°C for 30 s, for 40 cycles; and the third stage was maintained at 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s.

Total RNA was extracted from Cg and purified according to the standard procedure (Gao et al., 2016 (link)). Total purified RNA was reverse-transcribed into cDNA by a Reverse Transcription Kit (k1622, Thermo Scientific, United States). The primer for RAGE was designed at https://www.ncbi.nlm.nih.gov/tools/primer-blast/ (forward primer: TGA​CCC​TGA​CCT​GTG​CCA​TC; reverse primer: CCT​CAT​CCT​CAT​GCC​CTA​CCT​C). RT-qPCR was performed on the ABI 7500 real-time fluorescent quantitative PCR instrument (United States) using SYBR Green (420A, Takara, Japan). Cycle threshold values of genes of interest were normalized to gene GAPDH (forward primer: CCC​AGC​TTA​GGT​TCA​TCA​GGT; reverse primer: TAC​GGC​CAA​ATC​CGT​TCA​CA).

Peripheral blood was harvested (for patients with sepsis, fasting peripheral blood was collected within 24 h of admission; for healthy subjects, fasting peripheral blood was harvested on the enrollment). Total RNA was extracted using TRIzol (Takara Bio, Dalian, China). A Nanodrop2000 device was then utilized for the testing of the RNA concentration. RNA was reverse transcribed into cDNA using the Mir‐X miR First‐Strand Synthesis Kit (Takara Bio) or the PrimeScript RT reagent kit with gDNA Eraser Kit (Takara Bio). Subsequently, RT‐qPCR was performed on an ABI 7500 quantitative PCR instrument with the SYBR Premix Ex Taq kits (Takara Bio). The PCR primers were displayed in Table 1. U6 was utilized as the loading control for miR‐126‐5p. GAPDH was the internal reference of the TRAF6 mRNA. The determination of relative gene levels was realized with the 2^‐ΔΔCT method.

Guided by the manufacturer’s instructions, hepatic cDNAs of IRS2/phosphatidylinositol-3-kinases catalytic subunit alpha (PIK3CA)/AKT were synthesized (Kits from TIANGEN, DP431, Beijing, China). Realtime PCR was performed with the Applied Biosystems (Thermo Scientific™, ABI7500, Waltham, MA, USA) using the SYBR^® Premix ExTaqTM II (Takara, RR820A, Dalian, China). The sequences of primers (synthesized by Shenggong Bioengineering, Shanghai, China) are shown in Table 2 and their effectiveness were verified by the BLAST function test on PubMed. The mRNA expression was normalized against the corresponding control gene β-actin. Relative quantitative method was used to calculate the expression levels of the three genes, and the relative multiple change of the target gene = 2^−ΔΔCT:

Total RNA was extracted from cell lines and tissues using TRIzol reagent (Invitrogen, USA), the RT Reagent Kit with gDNA Eraser (TaKaRa, Japan) and SYBR Premix ExTaq^TM (TaKaRa, Japan). The RNA concentrations were determined using a NanoDrop 2000 (Thermo). The qRT-PCR assays were performed in an ABI 7500 (Foster City, CA, USA) using SYBR Green (TaKaRa, Japan). The relative expression levels of target genes were normalized against the control, and fold changes were calculated through relative quantification (2^-ΔΔCt). The sequences of qPCR primers (Sangon, Shanghai, China) are listed in Table S3.