Total cellular RNA from MSCs was extracted with TRIzol reagent (Takara Biotechnology, Dalian, China), and used for reverse transcription to cDNA. cDNA was synthesized by using Prime Script RT Master Mix (Takara Biotechnology), according to the manufacturer’s instructions. mRNA levels of Hes-1, Hey1, HeyL, Sdf-1α, Cxcr4, G-Csf, and Csf3r were measured by quantitative PCR using SYBR Green Ex Taq™ (Takara) detected by an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The PCR protocol consisted of 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, and 60°C for 34 s, with a final dissociation of 95°C for 30 s. The relative transcript levels were calculated by using the 2−∆∆CT method, in which ∆CT is the difference between the threshold cycle for the gene of interest and that for glyceraldehyde-3-phosphate dehydrogenase. The results were normalized to untreated controls. Primer sequences are listed in Table 1 .
Sybr green ex taq
Manufactured by Takara Bio
Sourced in United States
SYBR Green Ex Taq is a ready-to-use real-time PCR master mix that contains SYBR Green I dye and Ex Taq HS DNA polymerase. It is designed for sensitive and reliable quantification of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Prism 7500, by Thermo Fisher Scientific (5 mentions)
Primescript rt reagent kit, by Takara Bio (4 mentions)
Rnaiso plus, by Takara Bio (4 mentions)
Primescript rt master mix, by Takara Bio (2 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol, by Takara Bio (1 mentions)
Quantitative real time rt pcr analysis kit, by Bio-Rad (1 mentions)
Amv first strand cdna synthesis kit, by Roche (1 mentions)
Stepone software v2, by Thermo Fisher Scientific (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Sulphanilamide, by Merck Group (1 mentions)
Random oligonucleotide primers, by Promega (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Poly adp ribose polymerase parp, by Santa Cruz Biotechnology (1 mentions)
β actin monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltertazolium bromide mtt, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
10 protocols using sybr green ex taq
1
Quantitative PCR Analysis of MSC Genes
2
RT-qPCR Analysis of Gene Expression
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Total RNA was isolated from cultured cells with RNAiso Plus (Takara, Dalian, China), and cDNA was synthesized with the PrimeScript RT Reagent Kit (Takara). Then, 2 μl of cDNA was used for real-time PCR reactions in a Prism 7500 real-time thermocycler (Applied BioSystems, Foster City, CA, USA) with SYBR Green Ex Taq (Takara) according to the manufacturer's instructions. The primer sequences are provided in Supplementary Table 10 . Each group was made in triplicate.
3
Quantitative Analysis of Autophagy-Related Genes
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All primers were synthesized by Sangon Biotech Ltd. (Shanghai, China). Also, β-actin was used as an endogenous control, and the sequences of the additional primes were as follows:BECN1, forward: 5′-GGGTCACCATCCAGGAAC-3′, reverse: 5′-CACCATCCTGGCGAG-3′; ATG5, forward: 5′-GCGGTTGAGGCTCAC-3′, reverse: 5′-GGATATTCCATGAGT-3′; LC3, forward: 5′- GCGCTTGCAGCT-3′, reverse: 5′- GTACACTTCGGAGA-3′; and ACTB, forward: 5′-CCACCATGTACCCAGGCATT-3′, reverse: 5′-AGGGTGTAAAACGCAGCTCA-3′. Total RNA was extracted from the lung tissues using TRIzol (Takara Biotechnology, Dalian, China). RNA was then reverse-transcribed to cDNA using Prime Script RT Master Mix (Takara Biotechnology). The relative quantitative levels of mRNA were measured using SYBR Green Ex Taq™ (Takara Biotechnology), and PCR was performed according to the following conditions: initial denaturation at 95°C for 10 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 32 seconds. The comparative transcript levels were analyzed using the cycle threshold (∆∆Ct) method.
4
SYBR Green-Based Real-Time PCR in Prism 7500
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Real-time PCRs in a Prism 7500 real-time thermocycler (Applied BioSystems, Foster City, CA, United States) were performed with SYBR Green Ex Taq (Takara), according to the manufacturer’s instructions. The PCR conditions were 95°C for 30 s, followed by 40 cycles of 95°C for 3 s and 60°C for 30 s. All experiments were performed in triplicate. Fold changes in expression were calculated using 2ΔΔCt. The primer sequences are provided in Supplementary Table S1 .
5
Quantitative Analysis of Gene Expression
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The total RNA was isolated from 50 blastocysts using TRIzol reagent, and quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed using a Quantitative Real-Time RT-PCR Analysis Kit (Bio-Rad, Munich, Germany). The complementary DNA was synthesized using 1 µg of RNA, oligo (dT) primers, and the AMV First Strand cDNA Synthesis Kit (ROCHE). The quantitative PCR amplification was performed using SYBR® Green EX Taq™ (TaKaRa) and an RG-6000 Real-Time PCR detection system (Corbett Research Co., Mortlake, Australia). The samples were run in triplicate. The relative gene expression was calculated using the comparative threshold cycle (Ct) method, with GAPDH as the reference gene. The thermocycling conditions were as follows: 95 °C for 10 min; followed by 35 cycles at 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s. Fluorescence was measured once. Primer sets for each gene are listed in Table 1 .
6
Quantitative Real-Time PCR Analysis
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Total RNA was isolated from cultured cells with RNAiso Plus (Takara, Dalian, China), and cDNA was synthesized with the PrimeScript RT Reagent Kit (Takara). Then, cDNA and SYBR Green Ex Taq (Takara) were used for real-time PCR in a Prism 7500 real-time thermocycler (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The primer sequences are provided in Supplementary Table 4 . Each group was analyzed in triplicate.
7
RNA Extraction and Real-Time PCR Analysis
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s, with RNAiso Plus (Takara, Dalian, China), and cDNA was synthesized with the PrimeScript RT Reagent Kit (Takara). Then, 2 ml of cDNA was used for real-time PCR reactions in a Prism 7500 real-time thermocycler (Applied BioSystems, FosterCity, CA, USA) with SYBR Green Ex Taq (Takara), according to the manufacturer’s instructions. The primer sequences are provided in Supplementary Table 5 .
8
Real-Time PCR Protocol for Gene Expression
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The real-time PCR was conducted using StepOne plus (Applied Biosystems, Carlsbad, CA, USA) with SYBR Green Ex Taq (TaKaRa, Dalian, China) in 20 μL of total reaction mixture. The primers used were the same as ordinary PCR. The following conditions were used for amplification: 95 °C for 30 s, 40 cycles at 95 °C for 5 s, and 60 °C for 30 s.
For melt curve analysis, real-time PCR products were denatured at 95 °C for 15 s and then annealed at 60 °C for 1 min to allow the correct annealing of the DNA duplexes. These two steps were followed by melting curve ranging from 60 to 95 °C with temperature increments of 0.3 °C every 10 s. The data of real-time PCR and melt curve analysis were processed using StepOne Software v2.3 (Applied Biosystems, Carlsbad, CA, USA).
For melt curve analysis, real-time PCR products were denatured at 95 °C for 15 s and then annealed at 60 °C for 1 min to allow the correct annealing of the DNA duplexes. These two steps were followed by melting curve ranging from 60 to 95 °C with temperature increments of 0.3 °C every 10 s. The data of real-time PCR and melt curve analysis were processed using StepOne Software v2.3 (Applied Biosystems, Carlsbad, CA, USA).
9
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated from cells with RNAIso Plus (Takara Biotechnology Co., Ltd.), and RNA (100 ng) was reverse transcribed into single stranded cDNA using a PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd.) in 10 µl reaction at 37 C for 15 min, 85°C for 5 min, 4°C for hold. Then, 2 µl cDNA was used for qPCR using a Prism 7500 real-time thermocycler (Applied Biosystems; Thermo Fisher Scientific, Inc.) using with SYBR Green Ex Taq (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. The reaction protocol was as follows: 30 sec at 95°C, followed by 40 cycles of 5 sec at 95°C and 34 sec at 60°C. Relative expression level of genes was calculated using the 2−∆∆Ct method (18 (link)). The sequences of the primers were as follows: FOXP3, forward 5′-CGAAGCTTATGCCCAACCCCAGGCCTG-3′, reverse 5′-CGGGATCCTCAGGGGCCAGGTGTAGGGTTG-3′; PDCD4, forward 5′-TGGATTAACTGTGCCAACCA-3′, reverse 5′-TCTCAAATGCCCTTTCATCC-3′; SQOR, forward 5′-CACTGGTGGCTGTGGTAT-3′, reverse 5′-CACCCACTTTCCTCTTCAT-3′; PGGHG, forward 5′-GGTGGTCTCAGGAGGATGGA-3, reverse 5′-GGTCGGGTCAGAAGGAAGC-3′; and GAPDH, forward 5′-GTCAAGGCTGAGAACGGGAA3′ and reverse 5′-AAATGAGCCCCAGCCTTCTC-3′. Each reaction was set up in triplicate. GAPDH was used as the internal control.
10
Investigating Cellular Responses to Inflammatory Stimuli
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Dulbecco's modified eagle's medium (DMEM), foetal bovine serum, penicillin and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY, USA). iNOS, HO-1, Nrf2, p-c-Jun N-terminal kinase 1/2 (pJNK1/2), JNK1/2, p-extracellular signal-regulated kinase (pERK), ERK, poly(ADP ribose)polymerase (PARP), β-actin monoclonal antibodies and peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Random oligonucleotide primers and M-MLV reverse transcriptase were purchased from Promega (Madison, WI, USA). SYBR green ex Taq were obtained from TaKaRa (Shiga, Japan). iNOS, GCLC, GCLM, NQO-1 and β-actin oligonucleotide primers were purchased from Bioneer (Seoul, Korea). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tertazolium bromide (MTT), sulphanilamide, aprotinin, leupeptin, phenylmethylsulfonyl fluoride (PMSF), DL-Dithiothreitol (DTT), NS-398, LPS (Escherichia coli, serotype 0111:B4), LPS (Salmonella enterica, serotype enteritidis), Triton X-100, and all other chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA).
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