Total RNA was prepared from cells by TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and then RNA were reverse transcribed into cDNA by PrimeScript® 1st strand cDNA synthesis kit (TaKaRa, Tokyo, Japan). SYBR® Green Real-time PCR Master Mix (Sigma, USA) was used to determine the mRNA level of indicated gene by 7900HT qRT-PCR system. GAPDH was served as an internal control. Relative mRNA levels of indicated gene were analyzed using the 2-ΔΔCtmethod.
Sybr green real time pcr master mix
Manufactured by Merck Group
Sourced in United States
SYBR® Green Real-time PCR Master Mix is a ready-to-use solution for quantitative real-time PCR applications. The mix contains SYBR® Green I dye, DNA polymerase, dNTPs, and necessary buffer components.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
Superscript 3 first strand synthesis kit, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
7900ht real time pcr system, by Thermo Fisher Scientific (1 mentions)
Primescript 1st strand cdna synthesis, by Takara Bio (1 mentions)
Stratagene mx3000p qpcr system, by Agilent Technologies (1 mentions)
7 protocols using sybr green real time pcr master mix
1
Quantifying Gene Expression by qRT-PCR
2
Quantitative Real-Time PCR Assay
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Total RNA was extracted from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA (1 µg) was treated with DNase I (Invitrogen; Thermo Fisher Scientific, Inc.) and cDNA was synthesized using the SuperScript III First-Strand Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR was performed in a 7900 HT real time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR Green Real-Time PCR Master Mix (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). PCR conditions were: Initial denaturation, 95°C, 30 sec; 40 cycles: (Step 1) 95°C, 5 sec; (Step 2) 58°C, 15 sec; (Step 3) 72°C, 10 sec. GAPDH was used as an internal control. Relative mRNA levels were calculated using the 2−ΔΔCq method (10 (link)). The sequences of primers are given in Table I .
3
Quantify Gene Expression via qPCR
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GPC1, HAS1, HAS2, HAS3, EXT1, EXT2, DEFB1, TJP1, and CLDN3 expression was quantified with SYBR® Green Real time PCR Master Mix (Sigma). Reactions were carried out as previously described (5 (link)). The 2−ΔCt method was used to calculate relative mRNA expression normalized to the housekeeping gene GAPDH. The 2−ΔΔCt method was used for calculating fold change in gene expression levels versus untreated controls. All the primers were synthesized by Sigma-Aldrich as previously described inSupplemental Table 1
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GPC1, HAS1, HAS2, HAS3, EXT1, EXT2, DEFB1, TJP1, and CLDN3 expression was quantified with SYBR® Green Real time PCR Master Mix (Sigma). Reactions were carried out as previously described (5 (link)). The 2−ΔCt method was used to calculate relative mRNA expression normalized to the housekeeping gene GAPDH. The 2−ΔΔCt method was used for calculating fold change in gene expression levels versus untreated controls. All the primers were synthesized by Sigma-Aldrich as previously described in
4
Quantifying mRNA Expression Using RT-qPCR
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Total RNA was extracted from target cells with the help of TRIzol reagent (Invitrogen). The reverse transcription of extracted RNAs into cDNA was performed with the help of Maxima First Strand cDNA Synthesis Kits (K1672; Thermo Fisher). The expression of mRNA was determined with an SYBR® Green Real-time PCR Master Mix (Sigma, St. Louis, MO, USA) using GAPDH as an endogenous control. All the results were processed and analyzed using the 2-ΔΔCt method.
5
Quantifying SOD2 mRNA in Glutamate-Treated HT22 Cells
6
RNA Extraction and qPCR Analysis
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Total RNA was extracted from target cells with the help of TRIzol reagent (Invitrogen). The reverse transcription of extracted RNAs into cDNA was performed with the help of Maxima First Strand cDNA Synthesis Kits (K1672; Thermo Fisher). The expression of mRNA was determined with an SYBR ® Green Real-time PCR Master Mix (Sigma, St. Louis, MO, USA) using GAPDH as an endogenous control. All the results were processed and analyzed using the 2 -ΔΔCt method.
7
RNA Extraction and qPCR Analysis
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Total RNA was extracted from target cells with the help of TRIzol reagent (Invitrogen). The reverse transcription of extracted RNAs into cDNA was performed with the help of Maxima First Strand cDNA Synthesis Kits (K1672; Thermo Fisher). The expression of mRNA was determined with an SYBR ® Green Real-time PCR Master Mix (Sigma, St. Louis, MO, USA) using GAPDH as an endogenous control. All the results were processed and analyzed using the 2 -ΔΔCt method.
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