To extract total RNA from MKN-45 cells, we applied the Trizol reagent to separate the whole RNA according to the manufacturer’s directions (GeneAll Biotechnology, Korea). Subsequently, the extracted RNA (1 μg) was used to synthesize cDNA using its particular kit (Biofact, Daejeon, South Korea) via BioRad T100 thermal cycler system. Then, to evaluate B7-H7, Caspase3-8-9, BCL-2, and BAX genes expression, StepOne Plus qRT-PCR Device (Applied Biosystems, Foster City, USA) was utilized to carry out real-time PCR. GAPDH was served as an internal control to assess expression of genes. Also, all reactions were triplicated. For qRT-PCR results, 2-∆∆Ct method was utilized to compare the downregulation or upregulation of targeted genes in transfected or treated cells with control cells (control is assumed as 1). Gene-specific primers sets are presented in Table 2 .
Trizol reagent
Manufactured by GeneAll
Sourced in Cameroon
Trizol reagent is a versatile and effective tool for the isolation and purification of RNA from a wide range of biological samples. It is a single-phase solution of phenol, guanidine isothiocyanate, and other proprietary components designed to facilitate the efficient extraction and separation of RNA from DNA and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrometer, by Thermo Fisher Scientific (4 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
M mlv reverse transcriptase, by Bioneer (3 mentions)
Sybr green master mix, by Biofact (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Greenstar qpcr master mix, by Bioneer (2 mentions)
Rotor gene q thermocycler, by Qiagen (2 mentions)
Sybr green master mix, by Ampliqon (2 mentions)
Cdna synthesis kit, by Biofact (2 mentions)
Revertra ace qpcr rt kit, by Toyobo (2 mentions)
Cfx96 real time system, by Bio-Rad (2 mentions)
Direct zol rna miniprep, by Zymo Research (2 mentions)
Cdna synthesis kit, by EURx (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
T100 thermal cycler system, by Bio-Rad (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Rotorgenetm3000, by Qiagen (1 mentions)
Cdna synthesis kit, by GeneAll (1 mentions)
Rotor gene 6000 system, by Roche (1 mentions)
35 protocols using trizol reagent
1
Extraction and Quantification of Gene Expression in MKN-45 Cells
2
SIRT1 Gene Expression Quantification
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RNA was extracted from PBMCs using Trizol reagent (GeneAll Biotechnology Ltd., Seoul, Korea) and reversely transcribed into cDNA using a commercial kit (Applied Biological Materials Inc.-ABM, Richmond, VA, Canada). Five microliters of cDNA were used in a total 20 µL PCR mix containing 12.5 µL of SYBR GREEN PCR Master Mix (Applied Biosystems™, MA, USA), 1.25 µL of each primer, and 5µL of water. The primer sets used to amplify SIRT1 cDNA were: forward 5′-CCC TCA AAG TAA GAC CAG TAG CA-3′ and reverse 5′-AGT CTC CAA GAA GCT CTA CAT CA-3′. Actin was denoted as the internal control. The actin primers were: forward 5′-CAA CCG CGA GAA GAT GAC CC-3′ and reverse 5′-GAG GCG TAC AGG GAT AGC AC-3′. Gene expression was quantified using QuantStudio 3 Real-Time PCR System Software (Applied Biosystems™, MA, USA). After initial denaturation for 10 min at 95 °C, the amplification was carried out for 40 cycles (95 °C for 5 s, 60 °C for 30 s). The experiments were performed in triplicate, and data were analyzed by using the 2−ΔΔCt method as relative quantification.
3
RT-qPCR Analysis of RNA Expression
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For RT-qPCR, total RNA was extracted from the eWAT and liver by using TRIzol reagent (GeneAll Biotechnology, Seoul, Republic of Korea) [20 (link)]. cDNA was synthesized with M-MLV reverse transcriptase (Bioneer, Daejeon, Republic of Korea) by using an mRNA and cDNA synthesis kit with poly (A) polymerase tailing (ABM Inc., Richmond, BC, Canada) for miR. Next, RT-qPCR was performed with a Rotor-Gene Q thermocycler (Qiagen, Hilden, Germany) by adding Greenstar qPCR Master Mix (Bioneer). Table S2 lists the primer sequences designed by Primer3 [24 (link)], and mRNA expression was normalized using β-actin as a reference control. The specific primers of miR-34a, miR-370, and RNU6 were purchased from ABM Inc., and the expression levels of miRs were normalized using the expression of RNU6 snRNA as a control. The mRNA and miR expression levels were calculated via the 2−∆∆Ct method for relative quantification [25 (link)]. They were then expressed as fold differences compared with those of the HF group.
4
Quantifying Gene Expression in Adipocytes
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RT-qPCR was measured as previously described [19 (link)]. Total RNA from the 3T3-L1 adipocytes was extracted using TRIzol reagent (GeneAll Biotechnology, Seoul, Korea), and the cDNA was synthesized from RNA extracted using M-MLV reverse transcriptase (Bioneer, Daejeon, Korea). Afterward, RT-qPCR was performed in a fluorescent thermal cycler (Rotor-GeneTM 3000, Corbett Research, Mortlake, NSW, Australia) using Universal SYBR® Green PCR Master Mix (Bioneer Co., Daejeon, Korea). The 2−ΔΔCt method was used as a relative quantification strategy, and target gene expression was normalized using β-actin as an endogenous control. Primers used for RT-qPCR analysis are shown in Table 1 .
5
Tissue RNA Isolation and qRT-PCR Analysis
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We isolated the total RNA from tissue specimens utilizing the TRIzol reagent (GeneAll, Korea) according to the manufacturer's protocol. The RNA samples were then reversely transcribed to cDNA using a PrimeScript RT reagent kit (Takara, Japan). Next, we measured the gene expression via a quantitative real‐time polymerase chain reaction (qRT‐PCR) with the SYBR‐Green Master Mix (Ampliqon, Denmark). Each sample was performed in triplicate. We considered GAPDH as the endogenous control. Gene expression levels were normalized to GAPDH by using the 2−ΔΔct method. Table 2 enlists the primer sequences.
6
Gene Expression Analysis in Lung Tissue
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Total mRNA was isolated from frozen lung tissue of all
groups using TRIzol Reagent (GeneAll, South Korea) while
cDNA template was synthesized using cDNA synthesis Kits
(GeneAll, South Korea) according to the manufacturer’s
instructions. Pgm1 was used as an internal control while the
other primers were designed using the Gene runner software
and NCBI BLAST tool. Real-time PCR was performed using
Corbett’s RT PCR (Qiagen, USA). The primer sequences are
shown in Table 1. Real-time PCR efficiency for all mentioned
genes in Table 1 was determined using the slope of linear
regression as described by Pfaffl (22 (link)). Five samples in each
group were run in duplicates.
groups using TRIzol Reagent (GeneAll, South Korea) while
cDNA template was synthesized using cDNA synthesis Kits
(GeneAll, South Korea) according to the manufacturer’s
instructions. Pgm1 was used as an internal control while the
other primers were designed using the Gene runner software
and NCBI BLAST tool. Real-time PCR was performed using
Corbett’s RT PCR (Qiagen, USA). The primer sequences are
shown in Table 1. Real-time PCR efficiency for all mentioned
genes in Table 1 was determined using the slope of linear
regression as described by Pfaffl (22 (link)). Five samples in each
group were run in duplicates.
7
RNA Expression Analysis of Apoptosis and Cancer Markers
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Total RNA was extracted using the TRIzol reagent (GeneAll Biotechnology, Seoul, South Korea). A spectrophotometer (Thermo Fisher Scientific, Lenexa, South Korea) was used to examine the purity and concentration of the extracted RNA. The complementary DNA (cDNA) was synthesized using a thermal cycler system and the AddScript cDNA Synthesis Kit (ADDBIO, South Korea). Using a StepOnePlusTM Real-Time PCR System (Thermo Fisher, MA, USA), the mRNA expression of CASP9, CASP3, CASP8, MMP2, CD44, CDK6, CDK4, CCND1, RAF1, MAP2K1, MET, SRC, and CD274 was studied. 18s was used as the housekeeping gene. Before the qRT-PCR, the pair primer sequences were blasted before using the NCBI website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) (Table 1(Tab. 1) ). The obtained ct values were analyzed using log10 2-∆∆Ct.
8
Kidney RNA Extraction and Gene Expression
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Total RNA was extracted from kidney samples using Trizol reagent (GeneAll Biotechnology, Korea) according to the manufacturer’s instructions. The concentration and purity of RNA were determined by a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, USA). Then complementary DNA (cDNA) was synthesized according to the protocol of cDNA synthesis kit (Parstous, Iran). Real-time PCR amplification was performed on Mic PCR system (BMS, Australia) using SYBR Green master mix (Ampliqon, Denmark), cDNA and primers. Primer sequences were as follows: SIRT1, 5′-GGTAGTTCCTCGGTGTCCT -3′ (forward) and 5′-ACCCAATAACAATGAGGAGGTC-3′ (reverse); NRF2, 5′-CACATCCAGACAGACACCAGT-3′ (forward) and 5′-CTACAAATGGGAATGTCTCTGC-3′ (reverse); GAPDH, 5′-AGGTTGTCTCCTGTGACTTC -3′ (forward) and 5′-CTGTTGCTGTAGCCATATTC-3′ (reverse). PCR conditions were 95℃ for 10 min followed by 40 cycles of 95 °C for 40 s, 62 °C for 25 s, and 72 °C for 15 s. Relative gene expression was calculated using ΔΔCT method. GAPDH gene was used as internal control [20 (link)].
9
Quantifying Ang-1 and Ang-2 Expression via Real-Time PCR
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The expression of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) was measured using a real-time PCR technique (Table 1 ). The samples obtained from different groups were immediately frozen in liquid nitrogen and stored at -196˚C. RNA extraction was done by Trizol Reagent (Cat no: 302-001; Gene All) and reverse-transcribed to cDNA using an RNase-free DNase synthetase kit (Cat no: PP-410 S; Jena Bioscience). The PCR reaction was done using Corbett Life Science (Rotor-Gene 6000) System and Fast Start SYBR Green Master (Roche). Real-time PCR amplifications were performed using the following program: denaturation of cDNA (1 cycle at 94˚C for 10 min), amplification (40 cycles at 95˚C for 10 s, 60˚C for 35 s, and 72˚C for 20 s), and melting curve analysis (1 cycle at 60 to 95˚C with 1˚C/seconds). The 2−ΔΔCT method was used to evaluate the quantity.
Primers list
| Gene | Forward | Reverse | Annealing (°C) |
|---|---|---|---|
| ang-1 | 5′-GCCACTTGAGAATTACATTGTGG-3′ | 5′-CGCGGATTTTATGCTCTAATCAACTG-3′ | 59 |
| ang-2 | 5′-GTCTCCCAGCTGACCAGTGGG-3′ | 5′-TACCACTTGATACCGTTGAAC-3′ | 59 |
| gapdh | 5′-CTCTAAGGCTGTGGGCAAGGTCAT-3′ | 5′-GAGATCCACCACCCTGTTGCTGTA-3′ | 59 |
10
Total RNA Extraction from Tissue
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For extraction of total RNA, the tissue samples were homogenized with liquid nitrogen and their RNA was extracted by Trizol reagent (Geneall). Then, the quality and quantity of extracted RNAs were assessed using a NanoDrop spectrometer (Thermo Scientific, USA). After extraction, obtained RNAs were eluted in 50 μL of RNase-free water and stored at − 80 °C.
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