Total RNA of both wild type and mazEF mutant strains were extracted as described (Dai et al., 2020 (link)) and then cDNA was amplified using SYBR Premix Ex TaqTM GC (TaKaRa). RT-qPCR assays were performed using the Mx3005TMP Real-time detection system (Agilent, United States) and groEL was used to normalize RNA input. Experiments were repeated three times. RT-qPCR primers are listed in Supplementary Table S1 .
Sybr premix ex taqtm gc
Manufactured by Takara Bio
Sourced in Japan
SYBR Premix Ex TaqTM GC is a ready-to-use real-time PCR master mix that contains SYBR Green I dye, Taq DNA polymerase, and all other necessary reagents for real-time PCR amplification and detection. It is designed for sensitive and accurate quantification of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
7900 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (2 mentions)
Mx3005tmp real time detection system, by Agilent Technologies (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Taqman gene expression assay probe, by Thermo Fisher Scientific (1 mentions)
Taqman fast universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Thermo Fisher Scientific (1 mentions)
Rnaprep pure bacteria kit, by Tiangen Biotech (1 mentions)
7 protocols using sybr premix ex taqtm gc
1
Comparative RT-qPCR Analysis of mazEF Mutant
2
RNA Extraction and qRT-PCR Analysis in HK-2 Cells
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HK-2 cells (1 × 105 cells/ml) were plated into 6-well plates, and after treatment as described above, total RNA was extracted from all of the HK-2 cells using TRIzol® reagent (Invitrogen) according to the manufacturer’s instructions. NanoDrop™ 2000 (Thermo Fisher Scientific Inc.) was used to detect the quality and quantity of the extracted RNA. Complementary DNA (cDNA) was reverse-transcribed from quantified RNA using a PrimeScript TM RT reagent kit (Takara, Dalian, China) before qRT–PCR was used for gene amplification using the SYBR Premix Ex TaqTM GC (Takara) protocol according to their manufacturer’s instructions on a 7900 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The cycling programme involved preliminary denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 94°C for 2 min, annealing at 60°C for 50 s, and elongation at 60°C for 60 s, followed by a final elongation step at 60°C for 5 min. Relative gene expression was measured using the 2−ΔΔCt method. The primers used are listed in Table 1 . The ratio for the mRNA of interest was normalized to GAPDH.
3
Quantitative RT-PCR Analysis of HNPCs
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Total RNA of HNPCs was extracted using TRIzol reagent (Invitrogen) and the RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was conducted using PrimeScriptTMRT reagent Kit with gDNA Eraser (Takara, Beijing, China). The resulting products were used for qRT-PCR amplification with SYBR Premix Ex TaqTM GC (Takara). GAPDH or U6 was used as the normalization control. The primers for qRT-PCR are listed in Table 1 . The fold changes of mRNA or miRNA were determined by the 2–ΔΔCT method.
4
Comprehensive Profiling of Rice Anther Transcriptome
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Total RNA was isolated from various rice tissues, including anthers at different developmental stages, using Trizol reagent (Invitrogen) as described by the supplier. An aliquot of 1 μg RNA per sample was used to synthesize cDNA, using a PrimeScript RT reagent Kit with gDNA eraser (Takara). Quantitative reverse-transcription real-time PCR (qRT-PCR) was performed on a Bio-Rad C1000 machine using Takara SYBR Premix Ex TaqTM GC with a standard two-step protocol, consisting of 95 °C for 30s followed by 40 cycles of 95 °C for 5s and 60 °C for 30s. The expression level of OsACTIN1 was used as an internal control, and a relative quantitation method (Δ cycle threshold) was used to quantify the relative expression level of target genes. Three biological replicates with three technique replicates each were included for statistical analysis and error range analysis. The GenBank accession numbers of the genes used in the qRT-PCR assay are DPW (Os03g0167600), CYP703A3 (Os08g0131100), CYP704B2 (Os03g0168600), TDR (Os02g0120500), TIP2 (Os01g0293100), and MTR1 (Os02g0491300) (see Supplementary Table S1 for primer sequences). RNA in situ hybridizations were performed as described by Li et al. (2006) (link). A 269 bp cDNA fragment of OsGPAT3 was used for making antisense and sense probes (Supplementary Table S1 ).
5
Quantification of PAX6 Variants by qPCR
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The total RNA was extracted from the cells, and the synthesized cDNA underwent quantitative PCR amplification with TaqMan Gene Expression Assay probes (Supplementary Table S7 ) and a Taqman® Fast Universal PCR Master Mix (Life Technologies). We also designed two types of PAX6 primers and probes, to distinguish the expression of the two variants of PAX6, as described in the Supplementary Information . For the SYBR green quantitative PCR, cDNA was applied to quantitative PCR amplification with designed primers (Supplementary Table S8 ) and SYBR Premix Ex TaqTM GC (TaKaRa Bio, Otsu, Japan). cDNAs from the islet cells (Primary Cell Co., Sapporo, Japan) and ocular tissue (SightLife) were used as the positive controls for the qRT-PCR. The final mRNA levels were normalized to the GAPDH levels.
6
Quantifying Gene Expression in THP-1 Cells
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Trizol reagent (Invitrogen) was used to separate the total RNA from THP-1 cells according to the manufacturer's specifications. Before the real-time quantitative polymerase chain reaction (RT-qPCR) was performed for gene amplification using the SYBR Premix Ex TaqTM GC (TaKaRa, Tokyo, Japan) protocol on a 7900 real-time PCR system (Applied Biosystems, Foster City, USA), the complementary DNA (cDNA) was synthesized from quantified RNA with the use of a PrimeScript TM RT reagent Kit. Relative gene expression was measured using the 2 -ΔΔCt method. The ratio for the mRNA of interest was normalized to GAPDH. The primers used in this study are as follows: LOX-1 forward: 5'-TTGCCTGGGATTAG-TAGTGACC-3'. reverse: 5'-GCTTGCTCTTGTGTTAG-GAGGT-3; SR-A forward: 5'-GCAGTGGGATCACTTTCA-CAA-3', reverse: 5'-AGCTGTCATTGAGCGAGCATC-3'; Notch1 forward 5'-GAGGCGTGGCAGACTATGC-3': reverse 5'-CTTGTACTCCGTCAGCGTGA-3'; PPARγ forward: 5'-GGGATCAGCTCCGTGGATCT-3' reverse: 5'-TGCACTTTGGTACTCTTGAAGTT-3'; CD36 forward: 5'-GGCTGTGACCGGAACTGTG-3', and reverse: 5'-AGGTCTCCAACTGGCATTAGAA-3'.
7
Transcript Analysis of JSD-1 Cells
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Cultures collected from the basal salt medium were used to extract purified RNA. Basal medium contained 2.0 g/L KNO 3 , 1.0 g/L MgSO 4 , 0.5 g/L KH 2 PO 4 , 0.5 g/L NaCl, 0.25 g/L CaCl 2 , 0.01 g/L FeSO 4 •7H 2 O, 20.0 g/L agar, with the addition of 10.0 g/L each carbon source [pectin, rice straw, xylan, carboxymethyl cellulose (CMC), xylose or lignin]. Cells were sampled on the fifth day and total RNAs of JSD-1 were obtained using an RNAprep Pure Bacteria Kit (TIANGEN). Primers used for quantitative real-time PCR (qRT-PCR) were designed by DNAMAN 6.0 and are listed in Table 1.
Reverse transcription PCR for cDNA was performed followed by qRT-PCR using the SYBR Green method [SYBR® Premix Ex Taq TM GC (TAKARA)] according to the manufacturer's instructions. Cycling parameters of qRT-PCR reactions were programmed as follows: an initial step of 30 s at 95 °C followed by 40 cycles consisting of denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s and extension at 72 °C for 15 s. Relative quantification was achieved with the 2 -ΔΔCt method. Technical triplicates were performed for each biological replicate and average values were used for quantification. Herein, 16S rRNA was used as an internal control and treatment adding pectin was regarded as a control to normalize the relative transcription of the analyzed gene.
Reverse transcription PCR for cDNA was performed followed by qRT-PCR using the SYBR Green method [SYBR® Premix Ex Taq TM GC (TAKARA)] according to the manufacturer's instructions. Cycling parameters of qRT-PCR reactions were programmed as follows: an initial step of 30 s at 95 °C followed by 40 cycles consisting of denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s and extension at 72 °C for 15 s. Relative quantification was achieved with the 2 -ΔΔCt method. Technical triplicates were performed for each biological replicate and average values were used for quantification. Herein, 16S rRNA was used as an internal control and treatment adding pectin was regarded as a control to normalize the relative transcription of the analyzed gene.
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