Abi stepone plus real time pcr detection system

Total cellular RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations, followed by a reverse transcription with cDNA synthesis kit (ThermoFisher, Rockford, IL, USA). cDNA was synthesized from 2 µg of total RNA using 1 µL of reverse transcriptase and 50 ng/mL oligo(dT).
For detection of mature miRNAs (miRNA-1285, miRNA-25 and miRNA-30d), 500 ng of total RNA, miRNA-specific stem–loop RT primers (RiboBio), and PrimeScript RT reagent Kit (Qiagen, Valencia, CA, USA) were used in reverse transcription. U6 small nuclear RNA (RNU6B) was used as an internal control to determine relative miRNA expression. Each qPCR was carried out in triplicate using SYBR Green PCR Master Mix (Applied Biosystems) at one cycle of 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min on ABISteponePlus Real Time PCR Detection System (Applied Biosystems, USA). PCR mix was: 1 µL template cDNA, 5 µL SYBR Green PCR Master Mix (Applied Biosystems, USA), 3 µL ddH₂O, 1 µL (200 nM) of forward and reverse primers mix. All expression values were normalized against the housekeeping gene β-actin or the U6 (ΔC_t = C_{t target gene} − C_{t β-actin/U6}). Relative expression levels were then calculated as ΔΔC_t = ΔC_{t MeHg} − ΔC_{t control}, and relative expression changes were calculated as 2^−ΔΔCt. PCR primer sequences are available in the Table 1.

Total RNA was extracted from the samples with TRIzol (Vazyme, Nanjin, China). In this study, we extracted untreated ovarian cancer (Ovcar-4), breast cancer (MDA-MB-231, BT-549), colorectal cancer (Lovo, SW480) and gastric cancer (MKN45) cells’ RNA to examine the basal expression level of GPX3 in these cells. After transfected GPX3 knockout adenovirus (shGPX3), GPX3 overexpression adenovirus (oeGPX3) and corresponding control (Ctrl) into breast cancer (MDA-MB-231, BT-549), colorectal cancer (Lovo, SW480), gastric cancer (MKN45), and ovarian cancer (Ovcar-4) cells for 72h, their RNA was extracted. Later, we examined the efficiency of adenovirus transfection in regulating GPX3 expression. The complementary DNA was synthesized using a PrimeScript RT reagent Kit (Takara Bio, Otsu, Japan), messenger RNA expression was examined by real-time polymerase chain reaction (RT-PCR) using FastStart Universal SYBR Green Master Mix (Takara Bio, Otsu, Japan) and performed in ABI StepOne Plus Real-time PCR Detection System (Applied Biosystems, Foster City, CA). PCR recycling condition: 95 °C, 5min; 95 °C for 10s, 60 °C for 30s, 40 cycles.
The expression level of GAPDH was simultaneously quantified as an internal standard control. The sequences of all primers (Sangon, Shanghai, China) used were as follows:

Total cellular RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer's recommendations, followed by a reverse transcription with cDNA synthesis kit (Thermo). cDNA was synthesized from 2 µg of total RNA using 1 µL of reverse transcriptase and 50 ng/mL oligo (dT). Each qPCR was carried out in triplicate using SYBR Green PCR Master Mix (Applied Biosystems, CA, USA) at one cycle of 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min on ABI Stepone Plus Real Time PCR Detection System (Applied Biosystems). PCR mix included 1 µL template cDNA, 5 µL SYBR Green PCR Master Mix (Applied Biosystems), 3 µL ddH₂O, 1 µL (200 nM) of forward and reverse primers mix. Fold changes in the expression of each gene were calculated by a comparative threshold cycle (Ct) method using the formula 2^−(ΔΔCt). PCR primer sequences are available in Table 1.

The quantitative real‐time PCR (qRT‐PCR) reaction was performed in 96‐well plates using 2.0X RealQ‐PCR Master Mix^® with SYBR Green (Ampliqon) on ABI StepOnePlus™ Real‐Time PCR Detection System (Applied Biosystems). All qRT‐PCR primers were designed by Allele ID 6 software (Premier Biosoft) and described in Table 1. Each reaction mixture consisted of 1 µL cDNA (10 ng), 10 µL 2X RealQ‐PCR Master Mix^®, 1 µL (10 pmol/µL) of both forward and reverse primers, and 7 µL of PCR‐grade water, equating to a final volume of 20 µL. The beta‐2 microglobulin (β2M) mRNA was used as the reference gene. β2M was selected as the reference gene according to previous research for identification of housekeeping control genes in colorectal cancer.¹⁹ The thermal profile of the reaction was performed using the following conditions: initial denaturation at 95°C for 15 minutes; followed by 40 cycles at 95°C for 15 seconds and 60°C for 60 seconds followed by melting curve stage assessment. The melting curve profile and agarose gel electrophoresis were performed to verify the specificity of primers and the authenticity of the PCR products.

Total RNAs were extracted using the RNeasy Kit (Takara Bio). The qRT-PCR was carried out using SYBR Green Premix Ex TaqTM II (Takara Bio) on a ABI StepOnePlus Real-Time PCR Detection System (Thermo Fisher Scientific). Results were normalized to the housekeeping gene GAPDH. Relative gene expression levels from different groups were calculated with the 2-ΔΔCT method and compared with the expression level of appropriate control cells.
Specific primer sequences for individual genes were as follows: CCL5 (forward: 5′-GTATTTCTACACCAGCAGCAAG-3′; reverse: 5′-TCTTGAACCCACTTCTTCTCTG-3′); CXCL9 (forward: 5′-AATCCCTCAAAGACCTCAAACA-3′; reverse: 5′-TCCCATTCTTTCATCAGCTTCT-3′); CXCL10 (forward: 5′-CAACTGCATCCATATCGATGAC-3′; reverse: 5′-GATTCCGGATTCAGACATCTCT-3′); PD-L1 (forward: 5′-TGAGCAAGTGATTCAGTTTGTG-3′; reverse: 5′-CATTTCCCTTCAAAAGCTGGTC-3′); iNOS (forward: 5′-GCCGAGTGCAAGCATGGAGAG-3′; reverse: 5′-GGCTGTGAGGTGAGGTTGAAGAAG-3′); CD86 (forward: 5′-ACGGAGTCAATGAAGATTTCCT-3′; reverse: 5′-GATTCGGCTTCTTGTGACATAC-3′); CD206 (forward: 5′-CCTATGAAAATTGGGCTTACGG-3′; reverse: 5′-CTGACAAATCCAGTTGTTGAGG-3′); Arg1 (forward: 5′-CATATCTGCCAAAGACATCGTG-3′; reverse: 5′-GACATCAAAGCTCAGGTGAATC-3′); and GAPDH (forward: 5′-GTATTTCTACACCAGCAGCAAG-3′; reverse: 5′-TCTTGAACCCACTTCTTCTCTG-3′).