Total RNA was isolated from whole embryos using the RNeasy Mini Kit, according to manufacturer’s instructions (QIAGEN) for processing of animal tissues (see also Lee-Liu et al., 2012 (link); Nakamura et al., 2016 (link)). The abundance of RNAs was determined using a LightCycler 480 and SYBR Green I Master Reagents (Roche). Relative expression levels of genes were determined using ΔΔCt.
Sybr green 1 master reagent
Manufactured by Roche
Sourced in Switzerland, Germany
SYBR Green I Master reagent is a premixed solution containing SYBR Green I dye, necessary reagents, and enzymes for real-time PCR. The core function of this product is to enable the detection and quantification of DNA sequences during real-time PCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480, by Roche (4 mentions)
Lightcycler 480 system, by Roche (4 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (3 mentions)
Dnase 1, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Ethanol, by Nacalai Tesque (2 mentions)
2 propanol, by Nacalai Tesque (2 mentions)
Chloroform, by Nacalai Tesque (2 mentions)
Purelink rna mini kit columns, by Thermo Fisher Scientific (2 mentions)
Prelink dnase, by Thermo Fisher Scientific (2 mentions)
Oligo dt primer, by Thermo Fisher Scientific (2 mentions)
Omniscript rt kit, by Qiagen (2 mentions)
High pure rna isolation kit, by Roche (2 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Lightcycler instrument, by Roche (1 mentions)
Direct zol rna miniprep, by Zymo Research (1 mentions)
Sepasol, by Nacalai Tesque (1 mentions)
Faststart essential dna probes master, by Roche (1 mentions)
15 protocols using sybr green 1 master reagent
1
Quantitative Analysis of Embryonic RNA
2
Quantitative RT-PCR Analysis of RNA
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Total RNA was extracted from HBMECs using Direct-zol RNA MiniPrep (Zymo Research) with on-column DNase digestion. First-strand cDNA was synthesized using SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). qRT-PCR was performed on LightCycler instruments (Roche) using SYBR Green I Master reagents (Roche) according to the manufacturer’s recommendations. Relative mRNA expression was normalized to a reference gene (GAPDH).
3
Quantitative RT-PCR analysis protocol
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RT-qPCR analysis were performed as described previously101 (link) with minor modification. Total RNA was isolated from purified or cultured cells by using Sepasol (Nacalai Tesque) and chloroform (Nacalai Tesque), precipitated by using 2-propanol (Nacalai Tesque), and washed with 75% (vol/vol) ethanol (Nacalai Tesque). RNA samples were incubated with DNase I (Invitrogen, Carlsbad, California, USA) to remove contaminating genomic DNA and then reverse-transcribed into cDNA (Superscript III reverse transcriptase, VIRO cDNA Synthesis Kit; Invitrogen).
Quantitative PCR analysis was performed by using a LightCycler 480 II (Roche, Basel, Switzerland) with FastStart Essential DNA Probes Master (Roche) or SYBR Green I Master reagents (Roche). Primer sequences were: Tnfα sense, 5′-CTGTAGCCCACGTCGTAGC-3′; Tnfα anti-sense, 5′-TTGAGATCCATGCCGTTG-3′; S100a8 sense, 5′-TCCTTGCGATGGTGATAAAA-3′; S100a8 anti-sense, 5′-GGCCAGAAGCTCTGCTACTC-3′; Pparγ sense, 5′-GAAAGACAACGGACAAATCACC-3′; Pparγ anti-sense, 5′-GGGGGTGATATGTTTGAACTTG-3′; Nrf1 sense, 5′-GCTCTCTGAGACGCTGCTTT-3′; Nrf1 anti-sense, 5′-GTGTTCAGTTTGGGTCACTCC-3′; Actb sense, 5′-AAGGCCAACCGTGAAAAGAT-3′; and Actb anti-sense, 5′-GTGGTACGACCAGAGGCATAC-3′.
Quantitative PCR analysis was performed by using a LightCycler 480 II (Roche, Basel, Switzerland) with FastStart Essential DNA Probes Master (Roche) or SYBR Green I Master reagents (Roche). Primer sequences were: Tnfα sense, 5′-CTGTAGCCCACGTCGTAGC-3′; Tnfα anti-sense, 5′-TTGAGATCCATGCCGTTG-3′; S100a8 sense, 5′-TCCTTGCGATGGTGATAAAA-3′; S100a8 anti-sense, 5′-GGCCAGAAGCTCTGCTACTC-3′; Pparγ sense, 5′-GAAAGACAACGGACAAATCACC-3′; Pparγ anti-sense, 5′-GGGGGTGATATGTTTGAACTTG-3′; Nrf1 sense, 5′-GCTCTCTGAGACGCTGCTTT-3′; Nrf1 anti-sense, 5′-GTGTTCAGTTTGGGTCACTCC-3′; Actb sense, 5′-AAGGCCAACCGTGAAAAGAT-3′; and Actb anti-sense, 5′-GTGGTACGACCAGAGGCATAC-3′.
4
Quantitative RT-PCR for Gene Expression Analysis
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Reverse transcription and quantitative PCR analysis were performed as described previously [30 (link)]. In brief, RNA from cell suspensions was isolated by using Sepazol (Nacalai Tesque) and chloroform (Nacalai Tesque). After precipitation with 2-propanol (Nacalai Tesque) and washing with 75% (vol/vol) ethanol (Nacalai Tesque), the residue was incubated with DNaseI (Thermo Fisher Scientific) and reverse-transcribed to cDNA (Superscript 3 reverse transcriptase, VIRO cDNA Synthesis Kit; Invitrogen).
Real-time PCR was performed by using the LightCycler 480 System II (Roche, Basel, Switzerland) and SYBR Green I Master reagents (Roche). Primer sequences were: 5′-ggggatggagaagctacagg-3′ (sense) and 5′-tccgcttcaaacagagtgc-3′ (anti-sense) for Alox15, 5′-gaaagacaacggacaaatcacc-3′ (sense) and 5′-gggggtgatatgtttgaacttg-3′ (anti-sense) for Pparg and 5′-aaggccaaccgtgaaaagat-3′ (sense) and 5′-gtggtacgaccagaggcatac-3′ (anti-sense) for Actb.
Real-time PCR was performed by using the LightCycler 480 System II (Roche, Basel, Switzerland) and SYBR Green I Master reagents (Roche). Primer sequences were: 5′-ggggatggagaagctacagg-3′ (sense) and 5′-tccgcttcaaacagagtgc-3′ (anti-sense) for Alox15, 5′-gaaagacaacggacaaatcacc-3′ (sense) and 5′-gggggtgatatgtttgaacttg-3′ (anti-sense) for Pparg and 5′-aaggccaaccgtgaaaagat-3′ (sense) and 5′-gtggtacgaccagaggcatac-3′ (anti-sense) for Actb.
5
Comprehensive RNA and Protein Analysis
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Total RNA was extracted via TRIzol (Life Technologies, Carlsbad, CA) or an isolation kit (mirVana™ miRNA Isolation Kit, Ambion, USA). Reverse transcription was performed from total RNA using a Transcriptor First Strand cDNA Synthesis Kit (Roche) following the manufacturer's instructions. The qPCR primer of Suz12 and 18S rRNA was purchased from TaKaRa (Ruizhen). Bulge-loop™ miRNA qRT-PCR Primer Sets (one RT primer and a pair of qPCR primers for each set) specific to miR-320a and U6 were designed by RiboBio. The qPCR reactions were performed on a Light Cycler 480 system (Roche) with SYBR Green I Master reagents (Roche).
Western blots were performed as described previously [41 (link)] using antibodies specific to Suz12 (1:100, Abcam), EZH2 (1:2000, Origene), EED (1:2000, (Millipore), β-catenin (1:7500, Abcam), E-cadherin (1:1000, Cell Signaling Technology), and MMP-9 (1:2000, Proteintech). ImageJ software was used to analyze the expression of target proteins by comparison to GAPDH (1:5000, HC301) or H3 (1:2000, Cell Signaling Technology).
Western blots were performed as described previously [41 (link)] using antibodies specific to Suz12 (1:100, Abcam), EZH2 (1:2000, Origene), EED (1:2000, (Millipore), β-catenin (1:7500, Abcam), E-cadherin (1:1000, Cell Signaling Technology), and MMP-9 (1:2000, Proteintech). ImageJ software was used to analyze the expression of target proteins by comparison to GAPDH (1:5000, HC301) or H3 (1:2000, Cell Signaling Technology).
6
SYBR Green qPCR and ChIP-qPCR Protocols
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qPCR was performed using a LightCycler 480 and SYBR Green I Master reagents (Roche). For RT-qPCR, relative expression levels of each gene to odc1 were calculated and then normalised to the control. For primer sequences for RT-qPCR and ChIP-qPCR, see the supplementary Materials and Methods .
7
Tissue-specific Gene Expression Analysis
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Samples were taken from shoots and roots of 7‐day‐old seedlings, and from flag leaf blade, flag leaf sheath, culm, young panicle and mature floret before anthesis. Total RNA was extracted using RNAiso PLUS reagent (Takara, Dojima, Japan), and 1 μg RNA was reverse‐transcribed by oligo (dT) primers using a reverse transcription kit (Promega, Madison, WI, USA) after digestion with RNase‐free DNaseI (Thermo Scientific, Vilnius, Lithuania). The RT‐qPCR assay was performed in triplicate with SYBR Green I Master reagent and the Light Cycler Nano system (Roche, Mannheim, Germany). OsActin was used as the internal control for normalization. The primers used are listed in Table S1 .
8
RNA Isolation and qPCR Analysis
9
RNA Isolation and qPCR Analysis
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For RNA isolation, mouse embryos were washed in ice-cold PBS and homogenized in Trizol reagent (Invitrogen). Upon addition of chloroform, total RNA was isolated from water phase and purified with PureLink RNA Mini Kit columns (Ambion) and PreLink DNase (Invitrogen). cDNA was reverse transcribed from isolated RNA with Omniscript RT kit (QIAGEN) using oligo-dT primers (Thermo Scientific). Quantitative PCR was performed with SYBR Green I Master reagent (Roche) using Lightcycler 480 system (Roche). The expression of target genes was calculated by E-method (Tellmann, 2006 ) and normalized to the housekeeping gene (β-actin). All primer sequences are listed in Table S1 .
10
Quantitative RT-PCR Analysis of Gene Expression
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RNA was prepared from cells using the High Pure RNA isolation kit (Roche) followed by cDNA synthesis using SuperScript III or IV reverse transcriptase (Invitrogen, Thermo Fisher Scientific) and oligo (dT)18 primers (Invitrogen, Thermo Fisher Scientific). The mRNA expression levels of DNMT3a, DNMT3b, TLR4, RNA-binding motif protein (RBM) 14, IL-8, and GAPDH were quantified by real-time PCR using SYBR Green I Master reagent (Roche) or KAPA SYBR Fast qPCR kit (NIPPON Genetics, Tokyo, Japan) on a LightCycler 480 system (Roche) or CFX Connect real-time PCR detection system (Bio-Rad, Hercules, CA, United States). The following oligonucleotide pairs (F, forward; R, reverse) were used as PCR primers:
DNMT3a F: 5′-GACAAGAATGCCACCAAAGC-3′
DNMT3a R: 5′-CGTCTCCGAACCACATGAC-3′
DNMT3b F: 5′-AGCTCTTACCTTACCATC-3′
DNMT3b R: 5′-CCATCCTGATACTCTGAA-3′
TLR4 F: 5′-AAGCCGAAAGGTGATTGTTG-3′
TLR4 R: 5′-CTGAGCAGGGTCTTCTCCAC-3′
RBM14 F: 5′-CCTACGGCACGGTCATGAG-3′
RBM14 R: 5′-CGACACATTGCCCACGAAAA-3′
IL-8 F: 5′-GTGCAGTTTTGCCAAGGAGT-3′
IL-8 R: 5′-CTCTGCACCCAGTTTTCCTT-3′
GAPDH F: 5′-TGAACGGGAAGCTCACTGG-3′
GAPDH R: 5′-TCCACCACCCTGTTGCTGTA-3′
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