Total RNA of six human UM cell lines (92.1, MEL202, MEL270, MEL290, OMM2.3 and OMM2.5) and seven UM tissue samples was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using the PrimeScript™ RT Reagent kit (Takara Biotechnology Co., Ltd.). qPCR was subsequently performed using the SYBR® Green qPCR kit (Takara Biotechnology Co., Ltd.), on the ViiA™ 7 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The following thermocycling conditions were used for PCR: Initial denaturation at 95°C for 30 sec; 40 cycles of 95°C for 5 sec and 60°C (annealing and extension) for 34 sec, according to the manufacturer's protocol (SYBR® Green qPCR kit; Takara Biotechnology Co.,Ltd.). The following primer sequences were used for PCR: SNHG7: Forward, 5′-TTGCTGGCGTCTCGGTTAAT-3′ and reverse, 5′-GGAAGTCCATCACAGGCGAA-3′; GAPDH: Forward, 5′-TGTTGCCATCAATGACCCCTT-3′ and reverse, 5′-CTCAGCCTTGACGGTGCCAT-3′; β-actin: Forward, 5′-CATGTACGTTGCTATCCAGGC-3′ and reverse, 5′-CTCCTTAATGTCACGCACGAT-3′; U1: Forward, 5′-GACGGGAAAAGATTGAGCGG-3′ and reverse, 5′-GCCACGAAGAGAGTCTTGAAGG-3′. The relative mRNA levels were calculated using the 2−ΔΔCq method (32 (link)) and normalized to the internal reference gene GAPDH.
Sybr green qpcr kit
Manufactured by Takara Bio
Sourced in Japan, China, United States, Germany, Switzerland
The SYBR Green qPCR Kit is a reagent system designed for quantitative real-time PCR (qPCR) analysis. The kit utilizes SYBR Green I, a fluorescent dye that binds to double-stranded DNA, to detect and quantify target DNA sequences. The kit provides the necessary components, including the SYBR Green I master mix, to perform qPCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (28 mentions)
Trizol, by Thermo Fisher Scientific (16 mentions)
Primescript rt reagent kit, by Takara Bio (12 mentions)
Primescript reverse transcriptase, by Takara Bio (7 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Trizol kit, by Thermo Fisher Scientific (5 mentions)
Applied biosystems 7500 fast real time pcr system, by Thermo Fisher Scientific (5 mentions)
Trizol reagent, by Takara Bio (4 mentions)
Rnaiso plus, by Takara Bio (4 mentions)
Trizol, by Takara Bio (3 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Hiscript q rt supermix for qpcr, by Vazyme (3 mentions)
Primescript 2 1st strand cdna synthesis kit, by Takara Bio (3 mentions)
Reverse transcription kit, by Takara Bio (3 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Hiscript 2 q rt supermix for qpcr, by Vazyme (2 mentions)
Cfx96 instrument, by Bio-Rad (2 mentions)
M mlv kit, by Thermo Fisher Scientific (2 mentions)
106 protocols using sybr green qpcr kit
1
Quantifying lncRNA Expression in Uveal Melanoma
2
Quantifying Gene Expression in Apple and Arabidopsis
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Total RNA was isolated using E.Z.N.A.® Plant RNA Kit (Omega Biotek, Norcross, GA, USA) according to the manufacturer instructions. RNA integrity was checked on a 1.2% agarose gel and RNA quantity was assessed with a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). First-strand cDNAs were synthesized from 2 μg of total RNA with SYBR Prime Script RT-PCR Kit II (TaKaRa, Tokyo, Japan).
Semi-quantitative RT-PCRs were performed to examine the transcript levels of MpCYS4 in apple and Arabidopsis. Apple Actin and Arabidopsis Actin were used as respective loading controls. Quantitative real-time PCR (qRT-PCR) was conducted on an iQ5.0 detection instrument (Bio-Rad) using SYBR Green qPCR kits (TaKaRa). Reactions were performed in triplicate with a volume of 20 μL, and apple EF-1α or Arabidopsis Actin was amplified as the appropriate internal control. The relative quantity of target gene transcript was determined by applying the 2−ΔΔCT method (Livak and Schmittgen, 2001 (link)). Primers are listed in TablesS1 , S2 . Each value was expressed as mean and standard deviation (SD) calculated from the result of three independent replicates.
Semi-quantitative RT-PCRs were performed to examine the transcript levels of MpCYS4 in apple and Arabidopsis. Apple Actin and Arabidopsis Actin were used as respective loading controls. Quantitative real-time PCR (qRT-PCR) was conducted on an iQ5.0 detection instrument (Bio-Rad) using SYBR Green qPCR kits (TaKaRa). Reactions were performed in triplicate with a volume of 20 μL, and apple EF-1α or Arabidopsis Actin was amplified as the appropriate internal control. The relative quantity of target gene transcript was determined by applying the 2−ΔΔCT method (Livak and Schmittgen, 2001 (link)). Primers are listed in Tables
3
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted with an RNA Isolater Total RNA Extraction Reagent (Vazyme Biotech Co., Ltd., Nanjing, China). Reverse transcription was performed using the HiScript II Q RT SuperMix for qPCR (Vazyme, Nanjing, China). The primer pairs used in this study are all listed in Table S1 . The qRT-PCR was performed on an ABI7500 instrument (Applied Biosystems, Foster, CA, USA) using SYBR Green qPCR kits (TaKaRa, Kusatsu, Japan). The PCR amplification conditions included an initial heat-denaturing step at 95 °C for 3 min, followed by 40 cycles of 30 s at 95 °C, 30 s at 58 °C, and 1 min at 72 °C. Relative mRNA levels were normalized to that of the internal reference genes. Each sample was divided into 3 biological replicates. The data were processed on the basis of the 2−ΔΔCT method.
4
Quantification of GmDGK Genes Expression
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This study started with the selection of the reference genes for the qRT-PCR; Actin11 (leaves) and EF1A (roots) were used as the reference genes, and the primers were designed for all GmDGKs. We used the Primer Premier 5 software to design all 24 primers as shown in Table S1 (Supplementary Materials) [55 (link)]. For expression analysis of the GmDGK gene under abiotic stresses, we performed RT-PCR on a Stratagene Mx3000P thermocycler (Agilent) using SYBR Green qPCR kits (Takara) with the following settings: 95 °C for 15 s; 40 cycles of 95 °C for 15 s; and annealing at 58 °C for 30 s. Each reaction was performed in triplicate, and three biological replicates were done in the growth chamber. The expression levels of each gene at different time stress points was calculated using the 2−ΔΔCt method for abiotic treatments and the 2ΔCt method for different tissues [56 (link)].
5
Quantifying MdPHT Expression Profiles
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To obtain expression profiles for MdPHTs in different tissues and in response to phosphorus and/or drought treatment, we performed quantitative real-time PCR (qRT-PCR) on an iQ5.0 instrument (Bio-Rad, USA), using SYBR Green qPCR kits (TaKaRa) according to the manufacturer's instructions. The Actin gene served as our standard. Gene-specific primers were designed for these amplifications (Table S1 ). All reactions included 10.0 μL of SYBR® Premix Ex Taq™ (TaKaRa), 1.0 μL of cDNA template, 0.4 μL of each specific primer, and 8.2 μL of ddH2O2, made up to a 20-μL volume. The PCR conditions involved an initial 95°C for 3 min; then 40 cycles of 95°C for 20 s, 56°C for 20 s, and 72°C for 20 s. Based on three separate RNA extracts from three biological replications samples, each qRT-PCR was conducted three times to minimize inherent errors. Means and standard deviations were calculated from the results of three biological replicates. The relative expression levels of all MdPHT genes were calculated by the 2−ΔΔCT method (Livak and Schmittgen, 2001 (link)).
6
Watermelon Transcriptional Analysis via qRT-PCR
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Total RNA was isolated using the total RNA Miniprep Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer’s protocol. Then, RNase-free DNase I was added in RNA solution to remove any contaminated gDNA. First-strand cDNA synthesis was carried out following the manufacturer’s procedure (ReverTra Ace qPCR-RT Kit, Toyobo, Japan). Primers were designed using Primer Premier 5.0 software (Table S2 ). The qRT-PCR was performed on an CFX96 instrument (Bio-Rad, Alfred Nobel Drive Hercules, CA, USA) using SYBR Green qPCR kits (TaKaRa, Japan). The watermelon constitutive actin gene (Cla007792) was used as the endogenous control (Zhou et al., 2018b (link)). The PCR amplification conditions included an initial heat-denaturing step at 95 °C for 3 min, followed by 40 cycles of 30 s at 95 °C, 30 s at 58 °C, and 1 min at 72 °C. Relative expression levels were calculated using the 2−ΔΔCt method (Livak & Schmittgen, 2001 (link)), and each treatment included three independent biological replicates and three technical replicates. Data were statistically analyzed by one-way ANOVA using SPSS 19.0 software, and Tukey’s multiple range tests were used to detect significant treatment differences (P < 0.05).
7
Verification of Transcriptome Data by qRT-PCR
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To con rm the transcriptome data, the genes were randomly selected and veri ed and their primers were designed using the Primer Premier 5.0 software listed in Supplementary Table S5. Quantitative reverse-transcription PCR (RT-PCR) was performed on a Light Cycler® 96 instrument (Roche) using SYBR Green qPCR kits (TaKaRa) according to the manufacturer's protocol. Reactions were performed at 95°C for 2 min, 45 cycles of 95°C for 10 s, 58°C for 30 s and 72°C for 30 s. EF1α was used as the reference genes. The relative expression levels of the target genes were calculated using the 2 -△△CT approach.
8
Evaluating Gene Expression in Dermal Papilla Cells
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The SYBR Green qPCR kit (Takara, Dalian, China) was used to detect the mRNA expression levels of the above reverse transcription products. To further validate the effect of this gene of EGR1 on dermal papilla cells, we also used proliferation-related markers, including proliferating cell nuclear antigen (PCNA), and cyclin-dependent kinase 2 (CDK2). According to the sequences information in GenBank, primers were designed and synthesized by Qingke Biotechnology Co., Ltd. (Nanjing, China). We used the house-keeping gene (GAPDH) as an internal reference. All treatments included three biological replicates, and the primers information is shown in Table 2 .
9
Inflammatory Gene Expression in HaCaT Cells
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HaCaT cells (2 × 105) were seeded in 6-well plate and left to culture overnight. The cells were cultured in RPMI medium containing 2% (v/v) FBS and 10 μg/mL LPS, and varying soak solution mentioned above was added 1 hour later. After being treated for 6 h, RNA of HaCaT cells was isolated and purified with TriZol Reagent (Invitrogen) and chloroform. Reverse transcription of RNA into cDNA was performed with the cDNA synthesis kit (PrimeScript RT reagent kit, Takara, Japan), and then expression of genes encoding inflammatory-related factors, including TNF-α, IL-1β, IL-6, and p65(encoding genes were named as TNF-α, IL-1β, IL-6, and p65, respectively) was determined using a SYBR Green qPCR kit (Takara, Dalian, China) according to the manufacturer’s instructions and a previously described method,70 (link) with the GADPH as internal control. (The primer sequences are displayed in Appendix Table 1 ). The 2−ΔΔCT method was applied for relative quantitative analysis.
10
Quantifying Protein Expression in Cellular Samples
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Total RNA was extracted with TRIzol (Takara, Dalian, China), and the RNA quality was detected by the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., MA, USA). The cDNA was synthesized by M-MLV Kit (Invitrogen, Carlsbad, CA, USA). The SYBR Green qPCR kit (Takara, Dalian, China) was used to perform real-time quantitative PCR (RT-qPCR) in the Roche LightCycler 480 II system (Roche, Basle, Switzerland). The expression of the transcript is normalized by the expression level of β-actin.
RIPA lysis buffer (Beyotime, Shanghai, China) was used to extract the total protein of the sample, and the protein concentration was measured by a BCA assay (Takara, Dalian, China). The protein was separated by 10% SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to the PVDF membrane (Merck Millipore, MA, USA). Blocking with 5% skim milk in TBST for 1-2 hours, the PVDF membrane was incubated with primary antibodies overnight at 4°C. The primary antibodies were as follows: phospho-adenosine 5′-monophosphate- (AMP-) activated protein kinase (AMPK) α (Thr172) and AMPKα, phospho-acetyl-CoA carboxylase (ACC) (Ser79) and ACC, transforming growth factor- (TGF-) β1 (all from Cell Signaling Technology, Danvers, MA, USA), collagen I (Col-1) and fibronectin (FN) (both from Abcam, Cambridge, MA, USA), and α-smooth muscle actin (SMA) and β-actin (both from Boster Bio Co., Wuhan, China).
RIPA lysis buffer (Beyotime, Shanghai, China) was used to extract the total protein of the sample, and the protein concentration was measured by a BCA assay (Takara, Dalian, China). The protein was separated by 10% SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to the PVDF membrane (Merck Millipore, MA, USA). Blocking with 5% skim milk in TBST for 1-2 hours, the PVDF membrane was incubated with primary antibodies overnight at 4°C. The primary antibodies were as follows: phospho-adenosine 5′-monophosphate- (AMP-) activated protein kinase (AMPK) α (Thr172) and AMPKα, phospho-acetyl-CoA carboxylase (ACC) (Ser79) and ACC, transforming growth factor- (TGF-) β1 (all from Cell Signaling Technology, Danvers, MA, USA), collagen I (Col-1) and fibronectin (FN) (both from Abcam, Cambridge, MA, USA), and α-smooth muscle actin (SMA) and β-actin (both from Boster Bio Co., Wuhan, China).
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