The monolayer of vascular endothelial cells was washed twice with Ca2+- and Mg2+-free PBS and lysed with QIAzol lysis reagent (Qiagen). A quarter volume of chloroform was mixed with the lysate, centrifuged, and the supernatant was harvested, followed by addition of 70% ethanol at a concentration of 52.5%. The suspension was centrifuged at 20,000× g, and the supernatant was discarded. The precipitate was suspended again in 70% ethanol and centrifuged at 20,000× g, followed by collection and drying of the precipitate containing total RNA. Complementary DNA was synthesized from the mRNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster, CA, USA). Real-time PCR was performed using GeneAce SYBR qPCR mix α (Nippon Gene, Tokyo, Japan) with 1 ng/µL cDNA and 0.1 µM primers (Table 1 ) in a StepOnePlus real-time PCR system (Applied Biosystems). Levels of perlecan, syndecan-1, syndecan-2, syndecan-3, syndecan-4, glypican-1, biglycan, decorin, HIF-1α, HIF-2α, HIF-1β, β2-microgloblin (B2M), and β-actin mRNAs were quantified using the relative standard curve method. When the experimental group included Rh-Phen treatment or siRNA transfection, the fold change in intensity value of the target gene was normalized to that of B2M. In the other cases, the fold change in intensity value of the target gene was normalized to that of β-actin.
Geneace sybr qpcr mix α
Manufactured by Nippon Gene
Sourced in Japan, United States
GeneAce SYBR qPCR Mix α is a ready-to-use solution for performing quantitative real-time PCR (qPCR) reactions. It contains all the necessary components, including a DNA polymerase, SYBR Green I dye, and buffer system, optimized for efficient and reliable qPCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (9 mentions)
Qiazol lysis reagent, by Qiagen (4 mentions)
Bovine aortic endothelial cells, by Cell Applications (4 mentions)
Thermal cycler dice real time system, by Takara Bio (4 mentions)
Abi prism 7900ht, by Thermo Fisher Scientific (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Dulbecco s modified eagle s medium, by Nissui Pharmaceutical (2 mentions)
Calcium and magnesium free phosphate buffered saline, by Nissui Pharmaceutical (2 mentions)
Revertra ace qpcr rt master mix with gdna remover kit, by Toyobo (2 mentions)
Faststart sybr green master, by Roche (2 mentions)
Mouse monoclonal anti mt 1 2 antibody e9, by Agilent Technologies (2 mentions)
Anti β actin mouse monoclonal antibody, by Fujifilm (2 mentions)
Cmf pbs, by Nissui Pharmaceutical (2 mentions)
Isogen 2, by Nippon Gene (2 mentions)
Sepasol rna 1 super g, by Nacalai Tesque (2 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (2 mentions)
Cfx connect real time system, by Bio-Rad (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
29 protocols using geneace sybr qpcr mix α
1
Quantifying Endothelial Cell Gene Expression
2
Bovine Aortic Endothelial Cell Culture Protocol
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Bovine aortic endothelial cells were purchased from Cell Applications (San Diego, CA, USA). The following materials were purchased from the indicated vendors: Dulbecco's modified Eagle's medium and calcium-and magnesium-free phosphate buffered saline from Nissui Pharmaceutical (Tokyo, Japan); fetal bovine serum, Opti-MEM ® Reduced Serum Medium, Lipofectamine ® RNAiMAX Transfection Reagent, and High-Capacity cDNA Reverse Transcription Kit from Thermo Fisher Scientific (Waltham, MA, USA); cell culture dishes and plates from Corning (Corning, NY, USA); QIAzol lysis reagent from QIAGEN (Venlo, The Netherlands); GeneAce SYBR ® qPCR Mix α from Nippon Gene (Tokyo, Japan); and 1,3-diaminopropane from Tokyo Chemical Industry (Tokyo, Japan). Other reagents were purchased from Nacalai Tesque (Kyoto, Japan).
3
Real-time qRT-PCR for CD28 mRNA
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A real‐time quantitative reverse‐transcription polymerase chain reaction (RT‐PCR) assay for CD28 mRNA was carried out with a QuantStudio™ 12K Flex real‐time PCR system (Thermo Fisher Scientific) using GeneAce SYBR® qPCR Mix α (NIPPON GENE Co., Ltd.) according to the manufacturer’s instructions. Details are available in the supporting information file.34 , 35
4
Quantitative Gene Expression Analysis of Fungal Mycelium
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RNA isolation and cDNA synthesis were performed as described previously with slight modifications (15 (link)). Total RNA was isolated from frozen mycelial powder using Sepasol RNA I Super (Nacalai Tesque). One microgram of total RNA was then subjected to cDNA synthesis using the ReverTra Ace qPCR RT Master Mix with gDNA Remover kit (Toyobo). qRT-PCR assay was carried out using FastStart SYBR Green Master (Roche Applied Science) or GeneAce SYBR qPCR Mix α (Nippon Gene) according to the manufacturer’s instructions with specific primers for targets and an internal control gene (actin; MGG_03982). The primer sequences are given in Supplementary Table S1 . Fluorescence from DNA-SYBR Green complex was monitored by Thermal Cycler Dice Realtime System (Takara Bio) throughout the PCR reaction. The level of target mRNA, relative to the mean of the reference housekeeping gene was calculated by the comparative Ct method. Each experiment was performed with three technical replicates.
5
Quantifying mtDNA Levels in MELAS-iPSCs
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mtDNA copy numbers in MELAS-iPSCs and iPSC-derived cells were determined as described previously.8 (link) The reaction mixture contained 0.9 ng of template DNA and GeneAce SYBR qPCR mix α (Nippon Gene), with primers MT-CYB-F and MT-CYB-R for mtDNA or FBXO15-F and FBXO15-R for nuclear DNA (Table S1 ).
6
Quantitative RT-PCR for Gene Expression
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Quantitative RT-PCR was performed using an ABI Prism 7900HT instrument (Applied Biosystems) and GeneAce SYBR qPCR mix α (Nippon Gene, Japan). PCR cycling conditions included initial denaturation at 95°C for 10 min, followed by 45 cycles of 95°C for 30 s and 60°C for 1 min. ACTB was used as internal control. The primers used are listed in Table S1 .
7
Quantification of m.13513G>A mtDNA Heteroplasmy
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Quantification of m.13513G>A mtDNA heteroplasmy was performed by ARMS-qPCR using an ABI Prism 7900HT instrument (Applied Biosystems), as previously described.8 (link),36 (link) The reaction mixture contained 0.3 ng of template DNA and GeneAce SYBR qPCR mix α (Nippon Gene), with primers ARMS-G13513_F1WT and ARMS-G13513_R1 for wild-type mtDNA species or ARMS-G13513_F1Mut and ARMS-G13513_R1 for mutant mtDNA species (Table S1 ). qPCR was performed in triplicate.
8
Bovine Aortic Endothelial Cell Culture
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Bovine aortic endothelial cells were purchased from Cell Applications (San Diego, CA, USA). The following materials were purchased from the respective vendors: Dulbecco's modified Eagle's medium (DMEM) and calcium-and magnesium-free phosphate-buffered saline (CMF-PBS; Nissui Pharmaceutical, Tokyo, Japan); fetal bovine serum and a High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Waltham, MA, USA); QIAzol lysis reagent (QIAGEN, Valencia, CA, USA); GeneAce SYBR qPCR Mixα (Nippon Gene, Tokyo, Japan); mouse monoclonal anti-MT-1/2 antibody (E9; Dako, Glostrup, Denmark); Mouse monoclonal anti-β-actin antibody (Wako Pure Chemical Industries, Osaka, Japan); horseradish peroxidase-conjugated antimouse IgG antibody (#7076; Cell Signaling, Beverly, MA, USA); May-Grünwald and Giemsa stain solution (Merck KGaA, Darmstadt, Germany); and cadmium chloride, manganese chloride tetrahydrate, and other reagents (Nacalai Tesque, Kyoto, Japan).
9
Quantitative Real-Time PCR Protocol
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Large RNA from the cells which were extracted using Isogen II (Nippon gene) or TRIzol (Thermo Fisher Scientific) according to the manufacturer's protocol was used as the template. Extracted RNA was treated with RQ1 DNase I (Promega) at 37°C for 30 min. The RNA quality and quantity were determined by a NanoDrop 2000 (Thermo Fisher Scientific). Then, RNA (300 ng each) was reverse-transcribed using a SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). The reverse transcription mixture was incubated at 42°C for 60 min. The resultant cDNA solution was diluted with water by 1:5, and 1 µL of this solution was added to the real-time PCR mixture containing 5 µL of 2× GeneAce SYBR qPCR Mix α (Nippon gene), 0.5 µL of each primer in 10 µM, and 3 µL of H2O. 7900HT Real-Time PCR System (Thermo Fisher Scientific) was used. The cycling conditions were as follows: initial denaturation for 10 min at 95°C, 40 cycles of 15 sec at 95°C, 30 sec at 60°C. Fluorescence signals were collected at the 60°C step of each cycle. All reactions were run in duplicate and the Ct values were determined. The expression levels of target genes were normalized to the expression levels of ActB. To evaluate the reaction using the ΔCt method, we used a primer set shown in Supplemental Table S4 .
10
Quantitative Analysis of PAI-1 Promoter Binding
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ChIP assay was performed as previously described54 (link)60 (link). The purified DNA was analyzed by quantitative real-time PCR or semi-quantitative PCR. Quantitative real-time PCR was performed using GeneAce SYBR qPCR Mix α (NIPPON GENE) and a 7300 Real-Time PCR System (Applied Biosystems). For real-time PCR amplification, the following primer sequences were used: human PAI-1 promoter (SBE), 5′-GCAGGACATCCGGGAGAGA-3′ (forward) and 5′-CCAATAGCCTTGGCCTGAGA-3′ (reverse)67 (link); human PAI-1 promoter (p53RE/TSS), 5′-CCAAGAGCGCTGTCAAGAAGA-3′ and 5′-AGGAATTCAGCTGCTGGAGG-3′ (reverse)68 (link); and human HPRT1 first intron, 5′-TGTTTGGGCTATTTACTAGTTG-3′ (forward) and 5′-ATAAAATGACTTAAGCCCAGAG-3′ (reverse)54 (link). For semi-quantitative PCR amplification, human PAI-1 promoter (SBE), 5′-CCTCCAACCTCAGCCAGACAAG-3′ (forward) and 5′-CCCAGCCCAACAGCCACAG-3′ (reverse)69 (link) primers were used.
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