Total RNA was extracted from the larvae and pupae using the Trizol reagent (O10820, TransGen Biotech, Beijing, China) and then detected the RNA concentration, quantified 2 μg RNA transcribed to single stranded cDNA using a cDNA synthesis kit (G492, Abm, Richmond, Canada). qRT-PCR was performed in a final volume of 10 μL, containing 5 μL of TransStart Tip Green qPCR Supermix (291829AX, Aidlab, Beijing, China), 1 μL of cDNA (1:7 dilution), and 2 μL each of the forward and reverse primers (1 μM) (sequences showed in S1 Table ), the β-actin gene (Actb) was used as the internal reference. Data were analyzed by the formula R = 2– (ΔCt sample–ΔCt control), representing the relative transcription levels, ΔCt is the difference between the cycle threshold (Ct) of the genes and the average Actb transcript levels in the experimental or control sample.
Cdna synthesis kit
Manufactured by Applied Biological Materials
Sourced in Canada
The cDNA Synthesis Kit is a laboratory product designed to convert RNA into complementary DNA (cDNA) molecules. The kit provides the necessary reagents and enzymes to perform this conversion process, which is a fundamental step in various molecular biology and genetic research applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Formic acid, by Thermo Fisher Scientific (3 mentions)
Taq polymerase, by Thermo Fisher Scientific (2 mentions)
Pcr primers, by Macrogen (2 mentions)
Lightcyclerr 480 system, by Roche (2 mentions)
Dna ladder, by Thermo Fisher Scientific (2 mentions)
Pcr master mix, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Transstart tip green qpcr supermix, by Aidlab (1 mentions)
Trizol reagent, by Transgene (1 mentions)
Evagreen, by Applied Biological Materials (1 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Formaldehyde, by Avantor (1 mentions)
Thioflavin s stain, by Merck Group (1 mentions)
Tris edta, by Merck Group (1 mentions)
Entellan, by Electron Microscopy Sciences (1 mentions)
Triton x 100, by Duksan Pure Chemicals (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Diethyl ether, by Merck Group (1 mentions)
Paraffin wax, by Bio-Optica (1 mentions)
16 protocols using cdna synthesis kit
1
Quantification of Larval and Pupal Transcripts
2
Quantitative RT-PCR Analysis of Cardiac Gene Expression
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Total RNA in the left ventricular myocardium samples was extracted using the TRIZOL reagent (Thermo Fisher Scientific, Waltham, MA). After RNA quantification and purity evaluation, reverse transcription was conducted with a cDNA Synthesis Kit (abm, Richmond, BC, Canada), in a 20 μl reaction system containing 5× All-in-one, DEPC water, and RNA samples. Then, qPCR was performed with the EvaGreen (abm, Richmond, BC, Canada) on the Roche Light Cycler R480 System (Roche, Bedford, MA). The amplification conditions were: Pre-denaturation at 93°C for 10 s, denaturation at 55°C for 15 s, and annealing at 72°C for 20 s for 40 cycles. Three replicates were used for each sample. Gene expression were analyzed by using the 2−ΔΔCT method and normalized to β-tubulin. The sequences of primers (Sangon Biotech, Shanghai, China) are listed in Table 1 .
3
Quantifying MICALL2 mRNA Expression
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Total RNA was extracted from frozen tissue using TRIzol reagent and reverse transcribed into cDNA using a cDNA synthesis kit (Abm, Milton, ON, Canada). Real-time PCR analysis was performed with ABI 7500 real-time PCR system to specifically assess the relative abundances of MICALL2 mRNAs. The MICALL2 and 18S rRNA primers were as follows: MICALL2, 5’-AGTGACATCGTGGACTCGCT-3’ and 5’- TGGAGGCCCAGCTTCTCAATC-3’; 18S rRNA, 5’- GAAACGGCTACCACATCC-3’ and 5’-ACCAGACTTGCCCTCCA-3’. The experiment was repeated three times, and the relative expression level of the target gene was normalized to the mean of 18S rRNA. The data were analyzed using the comparative threshold cycle (2−ΔΔCT) method.
4
Histochemical Analysis of Protein Aggregation
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Agarose, dimethyl sulfoxide, ethidium bromide, hydrogen peroxide, paraformaldehyde, sucrose, Thioflavin-S- stain, Tris EDTA (Sigma Aldrich, St. Louis, MO, USA), biotin labeled 4G8 antibody (BioLegend, San Diego, CA, USA), boric acid (Serva, Heidelberg, Germany), DNA extraction kit (BioShop® Canada, Burlington, ON, Canada), cDNA synthesis kit (ABM, Vancouver, BC, Canada), DNA ladders (Invitrogen, Carlsbad, CA, USA), entellan (Electron Microscopy Sciences, Hatfield, PA, USA), ethanol, diethyl ether (Merck, Darmstadt, Germany), formaldehyde (BDH Chemicals Ltd, Poole, UK), 2X PCR master mix, Taq polymerase, skim milk, PCR grade water, formic acid (Thermo Fisher Scientific, Waltham, MA, USA), Imm PACT DAB, Elite ABC kit (Vector Laboratories, Burlingame, CA, USA), paraffin wax (Bio Optica Milano, Spa, Milan, Italy), PCR primers (Macrogen, Seoul, Korea), Superfrost* Plus microscope slides (Dako, Agilent, Santa Clara, CA, USA), TRI-reagent (BioShop® Canada, Burlington, ON, Canada), tris, tween 80 (Scharlau, Barcelona, Spain), and triton X-100 (Duksan, Kyungkido, Korea), and xylene (Lab Scan Ltd, Dublin, Ireland) were used in this study.
5
RNA and Protein Extraction from Shrimp Tissues
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TRIzol (ET101, Transgen, Beijing, China) was used for the extraction of total RNA from the hemocytes and different organs of shrimp. Proteins from the different organs were extracted using a radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% Nonidet P-40, 1 mM EDTA, and 0.5 mM PMSF, pH 8.0). The first-strand cDNA was synthesized using a cDNA synthesis kit (ABM, Vancouver, BC, Canada) according to the manufacturer’s instructions.
6
Quantitative RT-PCR Analysis of mRNA and miRNA
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Total RNA was extracted from cells using TRIzol reagent (Takara, Japan). Messenger RNA (mRNA) and miRNA were reverse-transcribed into complementary DNA with the cDNA Synthesis Kit (ABM, Vancouve, Canada). qRT-PCR was performed with a SYBR Green PCR kit (Takara, Kusatsu, Japan) and the Bio-Rad Real-Time PCR System (Bio-Rad, Hercules, CA, USA). GAPDH was treated as the internal control gene for HNRNPC, which U6 was the endogenous control gene for miRNAs. Primer information is presented in Table 1 . The relative expression level was analyzed using the 2−ΔΔCt. All assays were performed in triplicate.
7
Evaluating Serum-induced EMT Gene Expression
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Cells were seeded to 10 cm plates in 10% FBS and after they reach 70% confluency, they were overnight starved (incubated with serum-free RPMI) before adding 3% patient serum or control serum. RNA was isolated after 4 h of incubation using RNA isolation kit (Thermo Scientific, #K0732) quantified fluorometrically using RNA quantification system (QuantiFlour, Promega), and reverse transcribed via cDNA synthesis kit (ABM, #G234). For qPCR experiments, primers were designed using 2 different primer design tools (IBT technologies P-Quest and NCBI-BLAST) and listed as follows: SNAIL2 F: 5′- GCGATGCCCAGTCTAGAAA-3′ and R: 5′-GGTAATGTGTGGGTCCGAATA-3′; ZEB1 F: 5′- CTTCTCACACTCTGGGTCTTATTC-3′ and R: 5′- CGTTCTTCCGCTTCTCTCTTAC-3′; VIM F: 5′- CAGCTTTCAAGTGCCTTTCTG-3′ and R: 5′- CTTGTAGGAGTGTCGGTTGTT-3′; 18S F: 5′-CTTAGAGGGACAAGTGGCG-3′ and R: 5′-ACGCTGAGCCAGTCAGTGTA-3′ qPCR protocol was optimized according to 2X SYBR Green (Ampliqon RealQ Plus (2X) #A324402) protocol. 18S was used for internal control gene and 2^-( delta CT) calculations were performed to determine the fold differences between patient serum treated samples and controls.
8
Astaxanthin Mitigates AFB1-Induced Toxicity
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AFB1 (purity ≥ 98.0%, A606874) was purchased from Sangon Biotech (Shanghai, China) and dimethyl sulfoxide (DMSO) was obtained from Sigma Aldrich (St Louis, MO, USA). Fetal bovine serum (FBS) was supplied by Gibco (Gaithersburg, MD, USA). Astaxanthin (purity ≥ 98%) was obtained from Yuanye Bio-Technology (Shanghai, China). HyClone provided the DMEM/F12 and penicillin-streptomycin (Logan, UT, USA). Dojindo Laboratories offered the Cell Counting Kit-8 (CCK-8) for utilization in this research (Kumamoto, Japan). Kits for measuring ROS, annexin V, and propidium iodide (PI) were purchased from Beyotime Biotechnology (Shanghai, China). The assay kits of lactate dehydrogenase (LDH) and superoxide dismutase (SOD) were supplied by Jiancheng (Nanjing, China). The assay kits of malondialdehyde (MDA) and the glutathione (GSH) were purchased from Solarbio (Beijing, China). The reagents for real-time qPCR included BlasTaq 2X qPCR Mater Mix (ABM, Richmond, Canada) and cDNA synthesis kit (ABM, Richmond, Canada). The antibody against Nrf2 was provided by Abcam (Cambridge, MA, USA). Antibodies against Caspase-3, Caspase-9, cytochrome C (Cyt-C), heme oxygenase 1 (HO-1), Bax, Bcl-2, and GAPDH were supplied by Proteintech (Wuhan, China).
9
Gene Expression Analysis by qPCR in U87MG Cells
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Total RNA was extracted using RaPure Total RNA Micro Kit (Magen, R4111-03) according to the manufacturer’s instructions. Total RNA within 2 μg was reverse transcribed using cDNA Synthesis Kit (abm, G490). All interesting transcripts were quantified using SYBR qPCR enzyme (Vazyme, Q321−02) on CFX96 real-time system (Bio-Rad). Gene expression was normalized to GAPDH or vinculin. Primer sequences are listed in Supplementary Table 1 . siRNAs targeting STING and SEC23A were purchased from GenePharma and JTSBIO. Sub-confluent U87MG cells in 24-well plates were transfected with mixtures containing 20 nM siRNAs and 3 μl Lipofectamine RNAiMAX (Life Technologies, 13778150) or 1 μl lipid nanoparticles (LNP). 48 h posttransfection, the cells were used for downstream applications (viral infection). The siRNA sequences are listed in Supplementary Table 2 .
10
Quantifying Gene Expression in Lung Samples
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Frozen left lung samples (∼100 mg) were homogenized in TRIzol (Thermo Fisher Scientific, Waltham, MA, United States). The RNA was resuspended in nuclease-free water (20 μl) to later determination of its concentration and purity by spectrophotometry, being the 260/280 ratio of all samples in the range of 1.9–2.0. The RNA was stored at −80°C until later use. Synthesis to cDNA was performed using the cDNA Synthesis Kit (ABM, Richmond, Canada). The gene expression of TRPC1, TRPC4, TRPC6, ORAI1, ORAI2 and 18S was determined by real-time PCR (qPCR) in a StepOne Plus equipment (Applied Biosystems, Foster City, CA, United States), using a KicqStart kit (Sigma-Aldrich, United States) with the primers sequence used by Castillo-Galán et al. (2020) (link). Relative gene expression was calculated with the 2–ΔΔCt method using 18S gene.
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