Total RNA was isolated from BMDM or RAW264.7 cells using TRIzol RNA Isolation Reagents (TaKaRa, Janpan). Total RNA was reverse-transcribed into cDNA using a PrimeScript RT reagent Kit with gDNA Eraser (RR047A, TaKaRa, Japan). TB Green Premix Ex Taq II (RR820A, TaKaRa, Japan) was used for real-time fluorescent quantitative PCR (RT-PCR) to detect mRNA expression. ABI 7500 system (Applied Biosystems, United States) was used for performing real-time PCR. Primers sequences are in Supplementary information, Supplementary Table S1 . The final results were analyzed and calculated according to an equation: 2–ΔΔCt which provided the number of targets that were normalized to an internal reference. Ct value is the number of cycles required to reach the threshold during PCR process. The test was repeated three times for each biological sample.
Trizol rna isolation reagent
Manufactured by Takara Bio
Sourced in Japan, China
Trizol RNA isolation reagent is a mono-phasic solution of phenol and guanidine isothiocyanate that is used for the isolation and purification of total RNA from various biological samples, including cells, tissues, and microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit with gdna eraser, by Takara Bio (3 mentions)
Abi 7500 system, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Tb green premix ex taq 2, by Takara Bio (1 mentions)
Faststart universal sybr green master mix, by Roche (1 mentions)
Stepone system, by Thermo Fisher Scientific (1 mentions)
Tb green premix ex taqtm 2, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser perfect real time, by Takara Bio (1 mentions)
Primescript qrt pcr kit, by Takara Bio (1 mentions)
Sybr r primescript rt pcr kit, by Takara Bio (1 mentions)
Agilent 2000 bioanalyzer, by Agilent Technologies (1 mentions)
Human genome u133 plus 2.0 array, by Thermo Fisher Scientific (1 mentions)
Genespring gx software, by Agilent Technologies (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rt reagent kit, by Takara Bio (1 mentions)
Abi prism 7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Ab93876, by Abcam (1 mentions)
Ex taq dna polymerase, by Takara Bio (1 mentions)
12 protocols using trizol rna isolation reagent
1
Real-Time qPCR Analysis of Gene Expression
2
Quantitative Real-Time PCR for mRNA Expression
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mRNA expression was determined by quantitative real-time PCR (RT-qPCR). Total RNA from cells was isolated by TRizol RNA isolation reagents (Takara, Beijing, China) and one microgram of the total RNA was used to synthesize the first-strand cDNA using TaqMan reverse transcriptase kit (Takara Bio, Beijing, China). One microgram cDNA was subjected to RT-qPCR using FastStart Universal SYBR Green Master Mix (Roche, Switzerland) to detect the relative expression. Reactions were performed in triplicate. β-actin was used as internal control for normalization among cell samples. All the gene specific-primers used for RT-qPCR are listed in Table 1 .
3
Quantitative Real-Time PCR Analysis
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Total RNA was isolated from cells using TRIzol RNA Isolation Reagents (Takara, China). PrimeScriptTM RT reagent Kit with gDNA Eraser (RR047A, TaKaRa, China) was used for performing cDNA synthesis. mRNA was quantified with using TB Green Premix Ex TaqTM II (RR820A, TaKaRa, China). All experimental doses were in accordance with the manufacturer’s protocols. StepOneTM system (Thermo Fisher Scientific, Pittsburgh, PA, USA) was used for performing real-time PCR. The list of primers was as followed: TNF-α forward: 5’-AAGCCTGTAGCCCACGTCGTA-3’, reverse: 5’-GGCACCACTAGTTGGTTGTCTTTG-3’. IL-1β forward: 5’-GCAACTGTTCCTGAACTCAACT-3’, reverse: 5’-ATCTTTTGGGGTCCGTCAACT-3’. The final data were analyzed using Ct method and calculated and expressed using an equation (2–ΔΔCt), which provides the amount of the target that was normalized into an internal reference. Ct value represents the number of cycles required for fluorescence intensity to reach the threshold value in PCR process.
4
Quantitative RT-PCR Analysis of AMPK Expression
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Cell total RNA was extracted using Trizol RNA isolation reagent (Takara Bio Inc, Shiga, Japan). One μg of total RNA was reverse-transcripted into cDNA according to manufacturer instructions using a Takara RT kit. Real-time PCR was performed using a SYBR Premix Ex Taq II reagent kit (Takakra). The primer sets used were
5′-TGAAGATCGGCCACTACATCCT-3′,
5′-CACAGCAACTTTATGTCCAGTCAAC-3′ for AMPKα,
5′-TGACAGGATGCAGAAGGAGATTAC-3′,
5′-GAGCCACCAATCCACACAGA-3′ for beta-actin.
5
Total RNA Extraction from Liver Tissue
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Fifty milligrams of liver tissues (n = 4/time) were homogenized with 1 ml TRIzol® RNA isolation reagent (Takara, Japan) to extracted total RNA according to the manufacturer's guidance. Extracted RNA was resolved in 50 μl RNase-free water. Total RNA was checked by agarose gel electrophoresis, and then using a UV–Vis spectrophotometer (NanoDrop 2000c; Thermo Scientific, USA) to determine the quality of RNA. Additionally, the quantity and quality of RNA were estimated by NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). For gene cloning and real-time RT-PCR, 1 μg of total RNA was used for reverse transcription with a PrimeScript® RT reagent Kit with gDNA Eraser (Perfect Real Time; Takara) in a final reaction volume of 20 μl. The cDNA was stored at −20°C for later use.
6
Quantifying LOXL4 Expression in HCC
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Total RNA was isolated from HCC tissues and cell lines using TRIzol RNA isolation reagent (Takara, Japan) and reverse transcribed using a PrimeScript qRT-PCR kit (Takara, Japan) according to the manufacturer’s instructions. QRT-PCR was performed with an SYBR(R) PrimeScript RT-PCR Kit (Takara, Japan) using an ABI7500 system (Applied Biosystems, USA) and with specific primers: LOXL4 [forward 5’-CCGCTGCAAGTATGATGG-3′; reverse 5’-GTTCCTGAGACGCTGTTC-3′], 18sRNA [forward 5’-TGCGAGTACTCAACACCAACA-3′; reverse 5’-GCATATCTTCGGCCCACA-3′]. Relative LOXL4 gene expression analysis was performed using the eq. 2-ΔCt [ΔCt = Ct (LOXL4) – Ct (18sRNA)], with 18sRNA used as an internal control.
7
Hypoxia-Induced Transcriptome Changes
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HCT116 cells were treated with JMJD2B siRNA or control siRNA for 24 h, and then exposed to hypoxia for another 24 h. Total RNA was extracted from each sample using TRIzol RNA Isolation reagent (Takara) according to the manufacturer's instructions and purified with an RNeasy Mini Kit (Qiagen, Hilden, Germany). The quality and quantity of the purified RNA samples were evaluated using an Agilent 2000 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA). Data were collected with an Affymetrix Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA, USA) using standard methods and analysed using GeneSpring GX software (Agilent Technologies Inc.). Three biological replicates were tested. After normalisation, differentially expressed genes with statistical significance were identified through two compared groups (t-test, P<0.05). Relative changes in gene expression were calculated to identify genes that had been induced >2-fold and genes that had been downregulated by at least 0.5-fold after JMJD2B knockdown in hypoxia. Each up/downregulated gene was analysed for the relationship with DNA repair according to the published literature. Last, hierarchical clustering was performed to determine the extent of similarity between the up/downregulated genes.
8
Transcriptional Profiling of DNA Repair Genes
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Total RNA was extracted using TRIzol RNA Isolation reagent (Takara) and reverse-transcribed using an RT Reagent Kit (Takara) according to the manufacturer's protocol. Quantitative real-time RT–PCR (qRT–PCR) was performed using SYBR Premix Ex Taq II (Takara) at 95 °C for 30 s, and 40 cycles at 95 °C for 5 s and 60 °C for 30 s using an ABI Prism 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The qRT–PCR was carried out with the primers specific for JMJD2B and selected DNA repair-related genes: BMI1, CLU, CETN3, XPC, BTG2, and UNG (Supplementary Table 1 ). The qRT–PCR was performed in triplicate.
9
Quantitative Analysis of Stem Cell Differentiation
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RT-PCR, RT-qPCR, and western blot analysis were performed as described previously.40 (link) Total RNA was extracted from cultured cells using TRIzol RNA Isolation reagent (Takara, Tokyo, Japan), according to the manufacturer’s instruction. Three independent cultures were used for RNA preparations. First-strand cDNA was generated with High-Capacity cDNA Reverse Transcription Kit, (Applied Biosystems, San Diego, CA), and one microliter of each RT reaction mixture was amplified with Ex Taq DNA polymerase (Takara, Tokyo, Japan). As for RT-qPCR, cDNA was amplified using premix SYBR Green Ex Taq reagent kit (DRR820A, Takara) with a STEP ONE PLUS real-time PCR system (Applied Biosystems, Forster City, CA), according to the manufacturer’s instruction. All the primers used in this study are listed in Table 1 . As for western blotting, anti-Dlx2 (1:800; ab85995, Abcam, Cambridge, UK), anti-OCN (1:1 000; ab93876, Abcam), and anti-β-actin (1:3 000; EPR16769, Abcam, Cambridge, UK) were used for the detection of Dlx2, OCN, and β-actin, respectively. The secondary antibodies used this study were bought from Sigma-Aldrich and conjugated to horseradish peroxidase (anti-rabbit (1:5 000, A0545) or anti-mouse (1:5 000, SAB3701214)).
10
Extraction and Characterization of Fructan from C. pilosula
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The roots of C. pilosula Nannf. var. modesta L. T. Shen were collected in October 2017 from Jiuzhaigou County (Tibetan Qiang Autonomous Prefecture of Ngawa, China), and identified by Yuan-Feng Zou, College of Veterinary Medicine, Sichuan Agricultural University. The roots were dried and pulverized to a fine powder, and the fructan was obtained by DEAE-sepharose gel chromatograph, and identified as inulin-type fructan (the fructan form C. pilosula, CPPF) (Fu et al., 2018b (link)).
The cyclophosphamide (CY, C8650) was obtained from Solarbio technology Co., Ltd., (Beijing, China). The ELISA kits, including mouse IL-1β, tumor necrosis factor-α (TNF-α), sIgA and mucin 2 (Muc2), were purchased from Enzyme-linked Biotechnology Co., Ltd., (Shanghai, China). The acetic acid (71251), propionic acid (94425) and butyric acid (19215) standards were purchased from Sigma-Aldrich (St. Louis, MO, United States). The primeScript RT reagent kit (with gDNA Eraser, RR047A), the TB Green Premix Ex Taq II (Tli RNaseH Plus, RR821A) and Trizol RNA isolation reagent were obtained from TAKARA, Japan. All other chemicals, such as chloroform, isopropanol, etc., were of analytical grade, obtained from the Chengdu Kelong chemical factory (Chengdu, China).
The cyclophosphamide (CY, C8650) was obtained from Solarbio technology Co., Ltd., (Beijing, China). The ELISA kits, including mouse IL-1β, tumor necrosis factor-α (TNF-α), sIgA and mucin 2 (Muc2), were purchased from Enzyme-linked Biotechnology Co., Ltd., (Shanghai, China). The acetic acid (71251), propionic acid (94425) and butyric acid (19215) standards were purchased from Sigma-Aldrich (St. Louis, MO, United States). The primeScript RT reagent kit (with gDNA Eraser, RR047A), the TB Green Premix Ex Taq II (Tli RNaseH Plus, RR821A) and Trizol RNA isolation reagent were obtained from TAKARA, Japan. All other chemicals, such as chloroform, isopropanol, etc., were of analytical grade, obtained from the Chengdu Kelong chemical factory (Chengdu, China).
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