Q-PCR was carried out using SYBR® Premix Ex Taq™ system (Perfect Real Time, Takara) according to the manufacturer's protocol. Amplification and detection were run in a Sequence Detection System SLA-3296 (Bio-Rad) in triplicate with an initial cycle of 95°C for 10 seconds followed by 40 cycles of 95°C for 5 seconds, 60°C for 30 seconds, and 72°C for 20 seconds. Negative control samples (without template) were processed in the same way. The specificity of the amplifications was verified by melting curve analysis. The sequences of primers are as follows: Cadm1: forward primer GTG ATC CAG CTC CTG AAC CC, reverse primer CGT GTA GAG CTG GCA GAA GT and β-actin: forward primer CTC CAT CCT GGC CTC GCT GT, reverse primer GCT GTC ACC TTC ACC GTT CC. Relative quantization of Cadm1 expression normalized to β-actin was calculated according to the 2−ΔΔCT method.
Sybr premix ex taq system
Manufactured by Takara Bio
Sourced in Japan, United States, Germany, China
The SYBR Premix Ex Taq system is a ready-to-use solution for real-time PCR amplification. It contains a DNA polymerase, dNTPs, and a fluorescent dye SYBR Green I, which binds to double-stranded DNA and emits a fluorescent signal during the amplification process.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (26 mentions)
Primescript rt reagent kit, by Takara Bio (7 mentions)
Lightcycler, by Takara Bio (7 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Puromycin, by Merck Group (5 mentions)
Hexadimethrine bromide, by Merck Group (5 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (4 mentions)
Mission lentiviral packaging mix, by Merck Group (4 mentions)
Cfx96 real time pcr system, by Bio-Rad (4 mentions)
Doxorubicin, by Merck Group (4 mentions)
Moloney murine leukemia virus rt, by Thermo Fisher Scientific (3 mentions)
Sequence detection system sla 3296, by Bio-Rad (2 mentions)
Oligo dt 12 18, by Thermo Fisher Scientific (2 mentions)
M mlv reverse transcriptase, by Takara Bio (2 mentions)
Cfx connect real time pcr machine, by Bio-Rad (2 mentions)
Doxorubicin hydrochloride, by Merck Group (2 mentions)
Akr1c1 antibody, by Abnova (2 mentions)
Lamin b, by Santa Cruz Biotechnology (2 mentions)
Revertaid first strand dna synthesis rt kit, by Thermo Fisher Scientific (2 mentions)
Stratagene mx3005p detector, by Agilent Technologies (2 mentions)
64 protocols using sybr premix ex taq system
1
Real-Time qPCR Quantification of Cadm1 Expression
2
Quantitative PCR Validation of Novel Isoforms
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Real-time PCR was performed to detect selected transcripts using a SYBR Premix Ex Taq System (TaKaRa). PCR was performed to validate the novel isoforms by Q5 High-Fidelity DNA Polymerase (NEB). Primers are listed in the Supplementary Table.
3
IDO mRNA Expression in Breast Cancer
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The mRNA expression of IDO gene in breast cancer cell lines was analyzed using RT-PCR. Trizol Reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA, and MMLV reverse transcriptase (Promega, Madison, WI, USA) was used to reverse transcribe to cDNA. Expression levels of target genes were quantified using the SYBR Premix Ex Taq system (Takara Bio, Tokyo, Japan) following the manufacturer’s instructions. The primers applied in this assay were: IDO (188 bp), sense 5′-CATCTGCAAATCGTGACTAAG-3′; antisense 5′-CAGTCGACACATTAACCTTCCTTC-3′. β-actin (186 bp) was used as an internal control; sense 5′-TGGCACCCAGCACAATGAA-3′; antisense 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′. The specificity of the primers was verified and described in our prior papers (17 (link), 18 (link)). The thermal cycling program was listed below: initial denaturalization at 94°C for 5 min, then 94°C for 30 s, 58°C for 30 s, and 72°C for 45 s for 35 cycles; after the last cycle, 72°C for 10 min. All tests were repeated at least three times.
4
Quantification of Tmem64 mRNA Levels
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Total cellular RNA was extracted using the TRIzol-trichloromethane method, and RNA quantity was determined spectrophotometrically. Synthesis of cDNA was performed using the Takara PrimeScript® RT reagent Kit (Takara Bio). Quantitative PCR was performed using the SYBR Premix Ex Taq system (Takara Bio) and using the following primers: Tmem64 forward, 5′-GGCGTGGCTGAGGTGAGAAA-3′ and reverse, 5′-ATGAAGCCCACGACGAAGAG-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5′-CCAGTCAGCTTCCCGTTCA-3′ and reverse, 5′-GAACATCATCCCTGCATCCA-3′. The relative messenger RNA (mRNA) levels were calculated based on the threshold cycle (Cq) values normalized to the Cq value of β-actin using the following formula: 2−ΔCq, where ΔCq=Cqtarget gene-Cqβ-actin. All tests were performed in triplicate.
5
Quantitative RT-PCR Analysis of CD44 Expression
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Total RNA was isolated from the cells using the TRIzol reagent (Life Technologies). For the synthesis of cDNA, RT reactions were performed by incubating 200 ng of total RNA with a reaction mixture containing 0.5 μg/μL oligo dT12–18 and 200 U/μL moloney murine leukemia virus RT (Life Technologies). Real-time RT-PCR was carried out using a Roche Light Cycler (Mannheim, Germany) with the Takara SYBR Premix ExTaq System for relative quantification. Primers were synthesized by Bioneer (Daejeon, Republic of Korea) and primer sequences for the human genes are described in our previous studies [56 (link), 57 (link)]. PCR primers for CD44 are 5′-AGCAGCACTTCAGGAGGTTAC-3′ and 5′-TGCCTCTTGGTTGCTGTCTC-3′.
6
Quantitative RT-PCR Gene Expression Analysis
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Total RNA was extracted according to Zhao (2021) [31 ]. First-strand cDNA was synthesized from approximately 1 µg of total RNA using M-MLV Reverse Transcriptase (Takara Bio, Inc., Otsu, Japan). qRT-PCR was performed according to a standard protocol using a Bio-Rad CFX Connect Real-Time PCR machine (Bio-Rad Laboratories, Hercules, CA, United States) and the SYBR Premix Ex Taq™ system (Takara Bio, Inc.). The primers used are listed in Table S6 , and SiActin was used as an internal reference. The average value from at least three biological replicates is presented.
7
Antibody Validation for Cell Analysis
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The antibody against ALDH1A1 was obtained from Abcam (Cambridge, UK). Antibodies for NRF2, NQO1, BECN1, ATG7, lamin B, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies recognizing BCRP, KLF4, NANOG, c-Met, p62, LC3B, and MDR1 were purchased from Cell Signaling Technology (Danvers, MA, USA). AKR1C1 antibody was purchased from Abnova (Taipei City, Taiwan). The lentiviral expression plasmid for human NRF2 short hairpin RNA (shRNA), MISSIONTM Lentiviral Packaging Mix, hexadimethrine bromide, doxorubicin hydrochloride, ATRA, puromycin, and 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) were from Sigma-Aldrich (Saint Louis, MO, USA). The luciferase reporter plasmid for ARE was a gift from Dr. Wakabayashi (University of Pittsburg, PA, USA). The SYBR Premix ExTaq system was obtained from Takara (Otsu, Japan). Matrigel Basement Membrane Matrix was purchased from Corning Costar Corp (Cambridge, MA, USA).
8
Quantitative Real-Time RT-PCR Analysis
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Total RNA was isolated from clinical samples using TRIzol reagent (Invitrogen) according to the manufacturer’s recommended protocol. For quantitative real-time RT–PCR analysis, a Roche LightCycler was used with the Takara SYBR Premix ExTaq system. Primers were synthesized by Shanghai Sangon Biological Engineering Technology Services Co., Ltd. The nucleotide sequences of the primers were as follows: GAPDH, 5′-AAGGTGAAGGTCGGAGTCAA-3′ and 5′-AATGAAGGGGTCATTGATGG-3′; and HOXB7, 5′-ATCTACCCCTGGATGCGAAGCT-3′ and 5′-GCGTCAGGTAGCGATTGTAGTG-3′. Each sample was assessed in triplicate. Gene expression in the tumor cell lines or clinical samples was calculated relative to the GAPDH expression using the 2−ΔΔCt method.
9
Quantifying eIF3b Expression via RT-qPCR
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TRIzol Reagent (Invitrogen, Waltham, MA, USA) was used to extract the total RNA from cells and tissues, and the concentration and purity of the total RNA were detected by an ultraviolet spectrophotometer (Eppendorf, Germany). The RNA was reverse transcribed into cDNA with PrimeScriptTM RT Reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Japan). Real-time PCR was conducted using SYBR Premix Ex Taq System (Takara), using LightCycler® 2.0 Real-time PCR System (Roche, USA). The results were determined using the 2−ΔΔCt method. The primer sequences of eIF3b were as follows: 5′-CGGTGCCTTAGCGTTTGTG-3′ (forward) and 5′-CGGTCCTTGTTGTTCTTCTGC-3′ (reverse); the primer sequences of GAPDH were 5′-TGACTTCAACAGCGACACCCA-3′ (forward) and 5′-CACCCTGTTGCTGTAGCCAAA -3′ (reverse).
10
Comprehensive RNA Extraction and Analysis
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TRIzol (Invitrogen, Waltham, MA, USA) was used to extract total RNA from cells and tissues. The purity and quantity of total RNA were then detected by a NanoDrop Lite Spectrophotometer (Thermo Scientific, USA).
Nuclear and cytoplasmic RNAs were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, USA) according to the manufacturer’s protocol.
The RNA was reverse transcribed into cDNA with a Prime Script RT Reagent Kit (Takara Bio Inc, Osaka, Japan). qRT–PCR was conducted using a SYBR Premix Ex Taq System (Takara) and a CFX96 Real-time PCR System (Bio–Rad, USA). All of the primers are listed in Supplementary Table1 .
Nuclear and cytoplasmic RNAs were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, USA) according to the manufacturer’s protocol.
The RNA was reverse transcribed into cDNA with a Prime Script RT Reagent Kit (Takara Bio Inc, Osaka, Japan). qRT–PCR was conducted using a SYBR Premix Ex Taq System (Takara) and a CFX96 Real-time PCR System (Bio–Rad, USA). All of the primers are listed in Supplementary Table
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