Sybr green pcr assay

Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. The RNA was reverse transcribed into cDNA using the Prime Script RT Reagent Kit (Promega Corporation, Madison, WI, USA) with cDNA as the template. RT-PCR was performed using the SYBR green PCR assay (Takara, People's Republic of China). The primers for miR-100, U6, mTOR and GAPDH were purchased from Genechem Company (Genechem Co, Ltd, Shanghai, China). The PCR conditions consisted of 95 °C pre‑denaturation for 10 min, followed by 40 cycles at 95 °C for 5 s, 60 °C for 20 s, and extension at 72 °C for 15 s. qRT-PCR was performed on the Bio‑Rad CFX96/CFX Connect™ system (Bio‑Rad Laboratories, Inc, Hercules, CA, USA) to test the relative expression.

The total RNA was extracted from the cells using TRIzol reagent (TansGen biotech, Beijing, China), and the first-strand cDNA was synthesized by reverse transcriptase M-MLV (Takara, Beijing, China) with specific primers. The relative transcriptional level of IFN-β was detected by fluorescent quantitative real-time PCR (qPCR), using specific primers based on the human IFN-β sequence.
The above cDNA (1 μL of the 20 μL RT reaction mixture) was used as templates and subjected to SYBR green PCR assays (TaKaRa, Beijing, China) at least three times. qPCR was performed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), as follows: initial denaturation at 95 °C for 3 min followed by 40 cycles at 95 °C for 15 s, 56 °C. for 30 s, and 72 °C for 30 s. A final melting curve analysis was performed from 65 °C to 95 °C at a rate of 0.1 °C/s (continuous acquisition). The results are expressed as the relative gene expression level with normalization to the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative mRNA level was calculated using the 2^−ΔΔCt method.

RT-PCR was performed after 24 h incubation in the acidic conditions. RNA was extracted using TRIzol reagent (TAKARA, Dalian, China) as previously described [27 (link)]. Total RNA was reversely transcripted to cDNA utilizing a Double-Strand cDNA Synthesis Kit (TAKARA) according to the manufacturer's instructions. SYBR Green PCR assays (TAKARA) were used to perform real-time PCR in StepOnePlus (Applied Biosystems, USA). Three independent samples were set to ensure validity. 18S rRNA was used as internal control, and target genes were detected (Table 1). Primers were synthesized by Sangon Biotech (Shanghai, China), and quantitative real-time PCR data were calculated by the 2^−ΔΔCt method. PCR assays were conducted at least three times in triplicates for each sample.