RT-qPCR was used to assess the mRNA levels of acyl-coenzyme A cholesterol acyltransferase (ACAT1), monocyte chemoattractant protein 1 (MCP-1) and matrix metalloproteinase-9 (MMP-9) following the incubation of A7r5 cells with different treatments as aforementioned. Total RNA was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using the HiscriptTM First-strand cDNA Synthesis kit (Vazyme, Piscataway, NJ, USA) according to manufacturer's protocol for RT-qPCR analysis. The mRNA level of target genes was examined by RT-qRCR using SYBR Green I dye (Vazyme). The primers used for qRCR were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and their sequences were presented in Table I . The PCR assays were performed in triplicate on a Rotor-Gene 2000 Real-Time Quantitative PCR system (Corbett Life Science; Qiagen, Inc., Valencia, CA, USA). The amplification run was performed for 35 cycles under the following conditions: 95°C for 30 sec, 60°C (MCP-1)/60°C (MMP-9)/60°C (GAPDH) for 30 sec and 72°C for 30 sec. The semi-quantitative mRNA level of target gene to GAPDH was calculated using 2−∆∆Cq method and normalized with a media control (DMEM-F12 medium) (33 (link)).
Sybr green 1 dye
Manufactured by Vazyme
Sourced in China
SYBR Green I dye is a nucleic acid stain used in molecular biology applications, particularly in real-time PCR (qPCR) and other fluorescence-based techniques. It binds to double-stranded DNA (dsDNA) and emits green fluorescence upon excitation, allowing for the detection and quantification of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Hiscript first strand cdna synthesis kit, by Vazyme (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Cfx96, by Vazyme (1 mentions)
Oligo dt primer, by Sangon (1 mentions)
Hiscript 2 one step rt pcr kit, by Vazyme (1 mentions)
Rnaprep pure plant plus kit polysaccharides polyphenolics rich, by Tiangen Biotech (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
5 protocols using sybr green 1 dye
1
Quantifying mRNA Expression in A7r5 Cells
2
Quantifying Peroxisomal and IAA Genes in Rice
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The total RNA and cDNA were prepared as described above. To quantify the expression levels of genes related to peroxisomal H2O2 production and IAA metabolism in leaves of various GLO transgenic rice lines and WT, qRT-PCR analysis was performed on a Bio-Rad CFX96 apparatus with SYBR Green I dye (Vazyme). PCR was carried out in 96-well plates using the following program: denaturation for 5 min at 95 °C, followed by 40 cycles of denaturation for 10 s at 95 °C and incorporative annealing and extension for 30 s at 60 °C. The primers used for qRT-PCR were designed on a dedicated website (https://biodb.swu.edu.cn/qprimerdb/?tds-ourcetag=s_pcqq_aiomsg ). The data were normalized to the amplification of the OsActin1 gene (Os03g0718100). All experiments were performed with three biological and three technical replicates per biological replicate. The primer sequences used in this paper are presented in Supporting Information (Additional files 9 , 10 ).
3
Quantifying Inflammatory Gene Expression in HUVECs
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The cells were seeded at a density of 2×106 cells/well into a 6-well plate and starved in serum-free media for 12 h prior to stimulation. Total RNA was extracted from HUVECs using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using the HiScript™ First-strand cDNA Synthesis kit (Vazyme Biotech Co., Ltd.) at 50°C for 15 min and followed by 85°C for 2 min. oligo dT-primers (Sangon Biotech, Co., Ltd.) were used for the reverse transcription alongside 1 µg total RNA in a 10 µl reaction volume. The expression levels of target mRNAs in the HUVECs were subsequently analyzed via qPCR using SYBR-Green I dye (Vazyme Biotech Co., Ltd.). The primer pairs used for the qPCR are listed in Table SI . The following thermocycling conditions were used for the amplification run: Initial denaturation at 95°C for 30 sec; followed by 39 cycles at 56°C (VCAM-1)/58°C (ICAM-1, IL-1β and IL-6)/60°C [TNF-α, monocyte chemotactic protein (MCP-1), TLR4 and β-actin] for 30 sec and extension at 72°C for 30 sec. The expression levels of the target genes were quantified using the 2−Δ∆Cq method (28 (link)) and normalized to the control gene, β-actin.
4
Tissue-Specific Gene Expression Analysis
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Real-time quantitative RT-PCR was performed, as previously described by Li et al.35 (link). For tissue-specific expression analysis, total RNA was isolated from buds, stems, vascular bundles, first leaves, leaf veins of the first and third leaves, leaf veins of the third and fifth leaves, leaf veins of the fifth leaf, and roots using an RNAprep Pure Plant Plus Kit (polysaccharides & polyphenolics-rich) (TIANGEN, Beijing, China). The RNA concentration and integrity were evaluated via a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and confirmed via gel electrophoresis. Total RNA (1 μg) was reverse-transcribed with Oligo dT primer using a HiScript® II One Step RT-PCR Kit (Vazyme, China). qRT-PCR was performed on a Bio-Rad CFX96 in conjunction with SYBR Green I dye (Vazyme, China), while qPCR was performed on a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA). Primers were designed using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/ ). The results were normalized to those of a housekeeping gene, CsGAPDH, and calculated by the comparative Ct method36 (link). The primers used for real-time quantitative RT-PCR are listed in Supplementary Table S1.
5
RT-qPCR Analysis of Inflammation Markers
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Total RNA was extracted from cells using TRIzol® reagent (Invitrogen; USA). Complementary DNA (cDNA) was synthesized using the HiScriptTM First-strand cDNA Synthesis Kit (Vazyme, China). RT-qPCR was performed using SYBR Green I dye (Vazyme, China) and 10 ng cDNA. Briefly, after an initial denaturation step at 95°C for 30 s, the amplifications were carried out with 39 cycles at a melting temperature of 95°C for 30 s, an annealing temperature of 58°C (ICAM-1, IL-6, and β-actin)/54°C (IL-1β) for 30 s, and an extension temperature of 72°C for 30 s. The primer sequences are shown in Table S1 , and β-actin was selected as the housekeeping gene. The expressions of target genes were calculated using the 2-Δ∆Ct method.
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