RNA was extracted from GC and adjacent tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA using an RNA reverse transcription kit (Thermo Scientific, Waltham, MA, USA). We performed quantitative PCR using a qPCR kit (Takara SYBR Premix Ex Taq I, Tokyo, Japan) with primers (see Table S1 ) synthesized by Sangon Biotech (Shanghai, China). We used the 2−ΔΔCT method to analyze the data and normalized SKAP1 expression levels with the endogenous GAPDH mRNA level.
Rna reverse transcription kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, China
The RNA reverse transcription kit is a laboratory product that enables the conversion of RNA molecules into complementary DNA (cDNA) molecules. This process, known as reverse transcription, is a fundamental step in various molecular biology and genetic analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Cfx96 real time system, by Bio-Rad (15 mentions)
Trizol reagent, by Thermo Fisher Scientific (12 mentions)
Iq sybr green supermix, by Bio-Rad (9 mentions)
Rneasy mini kit, by Qiagen (8 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (4 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
C1000 thermal cycler, by Bio-Rad (3 mentions)
Plant total rna isolation kit, by Sangon (2 mentions)
Tri reagent, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix kit, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taq 1, by Sangon (1 mentions)
Fast real time pcr system, by Roche (1 mentions)
Premix ex taq, by Takara Bio (1 mentions)
Human gapdh, by Sangon (1 mentions)
Mouse β actin, by Sangon (1 mentions)
Temed, by Merck Group (1 mentions)
Pre stained protein ladder, by Thermo Fisher Scientific (1 mentions)
Cck 8 kit, by Dojindo Laboratories (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
47 protocols using rna reverse transcription kit
1
SKAP1 Expression Analysis in GC
2
Plant Total RNA Isolation and qPCR Analysis
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Total RNA was isolated from plant tissues using the Plant Total RNA Isolation Kit (Sangon, Shanghai, China), and the first-strand cDNA was synthesized by RNA Reverse Transcription Kit (Thermo Fisher, USA). The expression levels of genes were detected via quantitative real-time PCR (qPCR) using 26s rRNA as the reference gene. The reaction system and conditions were consulted with the manufacturer’s protocol [30 (link)]. Three biological replicates were conducted for each sample. The relative expression levels of genes were calculated using the 2-∆∆CT method. The gene-specific primers used for qPCR are listed in Table S1 .
3
Evaluating Tobacco Gene Expression Under Light Stress
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The materials from different plant parts were collected, including root. stem, the 3rd leaf, the 5th leaf, the 10th leaf, the 15th leaf, and flower at resettling stage, vigorous growing stage, and blooming stage, respectively. Four-week tobacco plants were selected for light treatments. The tobacco was treated with high light (1000 μmol m−2 s−1), and the leaves were collected after 1 h, 2 h, 4 h, and 6 h high light treatment. For low light treatment, the tobacco was treated by low light (100 μmol m−2 s−1), and the leaves were collected after 1 h, 2 h, 4 h, 6 h, and 8 h low light treatment. Total RNA was isolated from plant tissues using the Plant Total RNA Isolation Kit (Sangon, Shanghai, China), and the first-strand cDNA was synthesized by RNA Reverse Transcription Kit (Thermo Fisher, Waltham, MA, USA). The expression levels of genes were detected via quantitative real-time PCR (qPCR) using 26s rRNA as the reference gene. The reaction system and conditions were consulted with the manufacturer’s protocol [34 (link)]. Three biological replicates were conducted for each sample. The relative expression levels of genes were calculated using the 2−∆∆CT method. The gene-specific primers used for qPCR are listed in Table S2 .
4
Quantitative gene expression analysis in zebrafish
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Total RNA was extracted from 16 zebrafish embryos using Trizol reagent. Reverse transcription was performed with the Thermo Scientific RNA Reverse Transcription Kit. 2× PCR Mix (TaKaRa, Premix Ex Taq) containing SYBR Green I was used for the real-time quantitative PCR analysis with the Roche Applied Science Fast Real-Time PCR System. The corresponding gene primers are shown in Table S1 . The experiment was repeated at least three times.
5
Quantitative Real-Time PCR Analysis
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Total RNA was extracted from cells using TRIzol® reagent (cat. no. CW0580S; CoWin Biosciences) according to the manufacturer's instructions. Total RNA was quantified using a micro-spectrophotometer (NAno-300; cat. no. MO00040009; Hangzhou Allsheng Instruments Co., Ltd.), and the optical density 260/280 nm ratio of all samples was 1.8–2.0. Next, cDNA was synthesized on a PCR instrument (SimpliAmp™; cat. no. A24811; Thermo Fisher Scientific, Inc.) using the RNA reverse transcription kit (cat. no. CW2569M; CoWin Biosciences). RT was performed as follows: 42°C for 15 min, incubate at 85°C for 5 min, and keep warm at 4°C. Primers and a fluorescent quantitative PCR kit (cat. no. CW2601H; CoWin Biosciences) containing SYBR Green I fluorescent dye were used to perform fluorescent qPCR on a PCR system (cat. no. 4351106; Thermo Fisher Scientific, Inc.). Primer sequences for human HK2, human GAPDH, mouse HK2 and mouse β-actin (Sangon Biotech Co., Ltd.) are listed in Table II . The thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. The 2−ΔΔCq method was used for analysis of results and gene levels were normalized to the internal reference gene, GAPDH for human HK2 or β-actin for mouse HK2 (27 (link)).
6
Tripchlorolide Cytotoxicity Assay Protocol
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Tripchlorolide (T4) was purchased from Amresco (Amresco, CA, USA).CCK-8 kit was purchased from Dojin-do (Dojin- do, Japan), and dimethyl sulfoxide (DMSO), protease inhibitor cocktail and TEMED were purchased from Sigma (St. Louis, MO, USA). Protein quantitative BCA kit and RIPA lysate were purchased from Beyotime.30% acrylamide was purchased from Xiamen Lu Long Company and Ammonium persulfate (AP) from Amresco Company; Pre-stained Protein Ladder and RNA reverse transcription kit were purchased from Thermo Scientific. Western Blot chemiluminescence assay kit was purchased from KPL. High glucose medium, 1640 medium, penicillin and streptomycin were purchased from Hyclone and fetal bovine serum from GEMINI.
7
Oxidative Stress and Lipid Profile Analysis
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AMK was bought from Nanjing Tianyuan Dispensary (Nanjing, China). TRIzol reagent and AceQ qPCR SYBR Green Master Mix were from Vazyme (R401-01; H7901060; Vazyme, Nanjing, China). The RNA reverse transcription kit and PCR Mix were obtained from Thermo (K1622; Thermo, Waltham, MA). The Oil Red O Staining kit was purchased from Jiancheng Bioengineering Institute (D027-1-1; Jiancheng, Nanjing, China). Commercial test kits for malondialdehyde (MDA ) (A003-1-2), catalase (CAT ) (A007-1-1), glutathione peroxidase (GSH-Px ) (A005-1-2), total superoxide dismutase (T-SOD ) (A001-1-2), TC (A111-1-1), TG (A110-1-1), high-density lipoprotein cholesterol (HDL-C ) (A112-1-1), low-density lipoprotein cholesterol (LDL-C ) (A113-1-1), aspartate aminotransferase (AST ) (A010-2-1), alanine aminotransferase (ALT ) (A009-2-1) were all purchased from Nanjing Jiancheng Bioengineering Institute (Jiancheng, Nanjing, China). All other chemicals and reagents used were of analytical grade.
8
RNA Extraction and qPCR for Gene Expression
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Total RNA was extracted from the granulosa cells and 293T cells using Trizol following the manufacturer's protocol and quantified using the NanoDrop ND‐2000 spectrophotometer. Total RNA was reverse transcribed into cDNA using RNA reverse transcription kit (Thermo). For the detection of genes, qPCR was performed as previously described. The primers for genes are listed in Table 1 .
9
RT-PCR Analysis of Gene Expression
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The cells to be tested were inoculated into 6-well plates. Total RNA of the cells was extracted with TRIzol reagent (Sigma-Aldrich). CDNA was synthesized using RNA reverse transcription kit (ThermoFisher). Finally, using cDNA as template, the target gene was amplified by RT-PCR kit (Sigma-Aldrich). The temperature system is as follows: 94°C 45 s, 56°C 30s, 72°C 30s, 30 cycles, and at last 72°C extension for 10 min. MRNA levels of specific genes were detected by quantitative PCR instrument, and 3 multiple holes were set for miRNA detection of each sample to reduce errors.
10
Quantitative Analysis of Inflammatory Gene Expression
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Total RNA was used to synthesize cDNA using an RNA reverse transcription kit (Thermo Fisher, Waltham, MA) following the manufacturer’s protocol. Six inflammatory genes (IL-1α, IL-1β, IL-6, IL-10, Ccl3, and GM-CSF) and β-actin were quantified by quantitative real-time polymerase chain reaction (qPCR) using a LightCycler 96 (Roche, Basel, Switzerland). Reaction mixtures (10 μL) were amplified with 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. The following primers were used in this study: IL-1α, F: 5′-CCCATGATCTGGAAGAGACCA-3′, R: 5′-CAAACTTCTGCCTGACGAGC-3′; IL-1β, F: 5′-G CAGTGGTTCGAGGCCTAAT-3′, R: 5′-GCTGCTTCA GACACTTGCAC-3′; IL-6, F: 5′-CTCTCTGCAAGA GACTTCCATCC-3′, R: 5′-AAGTCTCCTCTCCGGAC TTGT-3′; IL-10, F: 5′-GGCGCTGTCATCGATTTC TC-3′, R: 5′-ATGGCCTTGTAGACACCTTGG-3′; Ccl3, F: 5′-TACAGCCGGAAGATTCCACG-3′, R: 5′-GTC AGGAAAATGACACCTGGC-3′; GM-CSF, F: 5′-CA GGGTCTACGGGGCAATTT-3′, R: 5′-ACAGTCCGT TTCCGGAGTTG-3′; β-actin, F: 5′-GATCAAGATC ATTGCTCCTCCTG-3′, R: 5′-AGGGTGTAAAACGC AGCTCA-3′. The 2−ΔΔ Cq method was used to calculate the relative mRNA levels.
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