mRNA transcription of cytokines was analyzed by qRT-PCR. Total RNA was extracted from BV2 microglial cells using TRIzol Reagent (Thermo Fisher Scientific) according to the manufacturer’s recommendations. Reverse transcription reaction was performed using EcoDry Premix Kit (TaKaRa, Tokyo, Japan). cDNA was subjected to qRT-PCR using SYBR Green Mix kit (Toyobo, Osaka, Japan) and the CFX Connect real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA). Primers, synthesized at Microgen (Daejon, Korea), were as follows: TNF-α: forward (F), 5′-GAT TAT GGC TCA GGG TCC AA-3′, reverse (R), 5′-GCT CCA GTG AAT TCG GAA AG-3′; IL1β: F’, 5′-AGC TGG AGA GTG TGG ATC CC-3′, R, 5′-CCT GTC TTG GCC GAG GAC TA-3′; IL-6: F′, 5′-CCA CTT CAC AAG TCG GAG GC-3′, and R′, 5′-GGA GAG CAT TGG AAA TTG GGG T-3′; GAPDH: F′, CAG GAG CGA GAC CCC ACT AA, and R′, ATC ACG CCA CAG CTT TCC AG. GAPDH is served as a normalization control. The relative RNA expression of each gene was analyzed using the 2−ΔΔCT method as previously reported [50 (link)].
Sybr green mix kit
Manufactured by Toyobo
Sourced in Japan
SYBR Green Mix kit is a ready-to-use solution designed for quantitative real-time PCR (qPCR) applications. It contains SYBR Green I, a fluorescent dye that binds to double-stranded DNA, enabling the detection and quantification of DNA amplification during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Cfx connect real time pcr system, by Bio-Rad (3 mentions)
Ecodry premix kit, by Takara Bio (2 mentions)
Revertra ace α reverse transcriptase kit, by Toyobo (2 mentions)
Revertaid reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time pcr detection system, by Toyobo (1 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (1 mentions)
Iq5 real time pcr system, by Bio-Rad (1 mentions)
5 protocols using sybr green mix kit
1
Cytokine mRNA Expression Analysis in BV2 Cells
2
Evaluating CHGX Extract's Impact on Candida albicans
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RNA extraction and qRT-PCR assay were performed, as described previously with a modification (Yue et al., 2018 (link)). Firstly, C. albicans yeast (SC5314) cells were initially cultured in liquid YPD medium at a logarithmic phase, and were then harvested, washed, and re-suspended in liquid Lee’s glucose medium. 2 × 106 cells/ml cells of SC5314 were treated with or without 20 mg/ml CHGX water-extract in Lee’s glucose medium at 30°C for 3 h. Cells were harvested for extraction of total RNA using the GeneJET RNA purification kit (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s instructions. For qRT-PCR assay, 1 μg of total RNA per sample was used to synthesize cDNA using the RevertAid Reverse Transcriptase kit (Thermo Fisher Scientific). Quantification of transcripts was carried out using a Bio-Rad CFX96 real-time PCR detection system with a SYBR green Mix kit (Toyobo Co., Ltd., Tokyo, Japan). The expression level of each protein was normalized to that of CaACT1.
3
Cytokine Expression Analysis by qRT-PCR
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mRNA transcription of cytokines was analyzed by qRT-PCR. Total RNA was extracted from SH-SY5Y cells using TRIzol Reagent (Thermo Fisher Scientific) according to the manufacturer’s recommendations. Reverse transcription reaction was performed using EcoDry Premix Kit (TaKaRa, Tokyo, Japan). cDNA was subjected to qRT-PCR using SYBR Green Mix kit (Toyobo, Osaka, Japan) and the CFX Connect real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA). Primers, synthesized at Microgen (Daejon, Korea), were as follows: for TNF-α, 5′-TCT CGA ACC CCG AGT GAC AA-3′ (sense) and 5′-TGA AGA GGA CCT GGG AGT AG-3′ (antisense); for IL-6, 5′-CAC AGA CAG CCA CTC ACC TC-3′ (sense) and 5′-TTT TCT GCC AGT GCC TCT TT-3′ (antisense); for β-actin, 5′-CTT CCT GGG CAT GGA GTC CT-3′ (sense) and 5′-GGA GCA ATG ATC TTG ATC TT-3’ (antisense). β-actin served as a normalization control. The relative RNA expression of each gene was analyzed using the 2−ΔΔCT method as previously reported [37 (link)].
4
Quantification of Gene Expression via RT-PCR
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The cells (2 × 104) were seeded in 24-well plates and treated with various concentrations of MSU crystals (0.1, 0.2, and 0.3 mg/mL) for 24 h. The total RNA was extracted using a TRIzol reagent, and complementary DNA (cDNA) was synthesized using a ReverTra Ace-α-reverse transcriptase kit (Toyobo, Osaka, Japan). The cDNA was then analyzed by real-time RT-PCR (Bio-Rad iQ5 Real-Time PCR System, Bio-Rad, Hercules, CA, USA) using an SYBR Green Mix kit (Toyobo, Osaka, Japan). The PCR amplification consisted of an initial denaturation at 95 °C for 15 min, followed by 40 cycles of 9 °C for 5 s, 55–65 °C for 30 s, and 72 °C for 15 s. The relative expression of each gene was analyzed using the ΔΔCT method.
5
Osteoclastogenesis Regulatory Genes Analysis
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Cells (2 × 104/well) were seeded in 24-well plates and pre-incubated with sRANKL (100 ng/ml) for 4 days and then cotreated with CpG-ODN (1 μM) for 2 days. Total RNA was extracted from cells using TRIzol Reagent (Gibco BRL, Grand Island, USA), and complementary DNA (cDNA) was synthesized using a ReverTra Ace-α-reverse transcriptase kit (Toyobo, Osaka, Japan). cDNA was subjected to real time PCR using SYBR Green Mix kit (Toyobo, Osaka, Japan) and the CFX Connect real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturers' instructions.
Primers, synthesized at Bionics Company (Seoul, Korea), were as follows: TRAP, forward 5′- AAG GCG AGA GAT TCT TTC CCT G-3′, reverse 5′-ACT GGG GAC AAT TCA CTA GAG C-3′; cathepsin K, forward 5′- CAG CAG AAC GGA GGC ATT GA-3′, reverse 5′-CCT TTG CCG TGG CGT TAT AC-3′; carbonic anhydrase II, forward 5′- CAT TAC TGT CAG CAG CGA GCA-3′, reverse 5′-GAC GCC AGT TGT CCA CCA TC-3′; NFATc1, forward 5′-CTC GAA AGA CAG CAC TGG AGC AT-3′, reverse 5′-CGG CTG CCT TCC GTC TCA TAG-3′; c-Fos, forward 5′-ACC ATG ATG TTC TCG GGT TTC AA-3′, reverse 5′-GCT GGT GGA GAT GGC TGT CAC-3′; A20, forward 5′-TGCCCAGTCTGTAGTCTTCG-3′, reverse 5′-AGTTGTTCAGCCATGGTCCT-3′; and GAPDH, forward 5′-TGC ACC ACC AAC TGC TTA-3′, reverse 5′- GGA TGC AGG GAT GAT GTT C-3′. The relative expression of each gene was analyzed using the 2–ΔΔCT method.
Primers, synthesized at Bionics Company (Seoul, Korea), were as follows: TRAP, forward 5′- AAG GCG AGA GAT TCT TTC CCT G-3′, reverse 5′-ACT GGG GAC AAT TCA CTA GAG C-3′; cathepsin K, forward 5′- CAG CAG AAC GGA GGC ATT GA-3′, reverse 5′-CCT TTG CCG TGG CGT TAT AC-3′; carbonic anhydrase II, forward 5′- CAT TAC TGT CAG CAG CGA GCA-3′, reverse 5′-GAC GCC AGT TGT CCA CCA TC-3′; NFATc1, forward 5′-CTC GAA AGA CAG CAC TGG AGC AT-3′, reverse 5′-CGG CTG CCT TCC GTC TCA TAG-3′; c-Fos, forward 5′-ACC ATG ATG TTC TCG GGT TTC AA-3′, reverse 5′-GCT GGT GGA GAT GGC TGT CAC-3′; A20, forward 5′-TGCCCAGTCTGTAGTCTTCG-3′, reverse 5′-AGTTGTTCAGCCATGGTCCT-3′; and GAPDH, forward 5′-TGC ACC ACC AAC TGC TTA-3′, reverse 5′- GGA TGC AGG GAT GAT GTT C-3′. The relative expression of each gene was analyzed using the 2–ΔΔCT method.
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