To examine the expression pattern of MviPBANR-B and -C in each developmental stage (eggs, 1st–5th instar larvae, pupae, and adults) and adult tissues (head, thorax, abdomen without pheromone gland or hair pencil, pheromone glands, and hair pencils), total RNA extraction and cDNA synthesis were performed. Quantitative real-time PCR (qRT-PCR) was performed with TOPreal™ qPCR 2× PreMIX (SYBR Green with low ROX) (Enzynomics, Republic of Korea) and specific primers (Table S1 ). The following parameters were used for the qRT-PCR: 95 °C for 10 min, followed by 50 cycles at 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. Experiments were repeated independently in triplicate and elongation factor 1-α(EF1-α) and ribosomal protein S18 (RPS18) were used as positive controls. The relative expression levels of MviPBANR-B and -C were calculated using the 2−ΔΔCT method [29 (link)].
Topreal qpcr 2 premix sybr green with low rox
Manufactured by Enzynomics
Sourced in United States
TOPreal™ qPCR 2× PreMIX (SYBR Green with low ROX) is a ready-to-use solution for real-time quantitative PCR (qPCR) assays. It contains SYBR Green I dye and low ROX reference dye, which are essential components for qPCR amplification and detection.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Improm 2 reverse transcriptase kit, by Promega (2 mentions)
Cfx96 real time system, by Bio-Rad (2 mentions)
Topscript cdna synthesis kit, by Enzynomics (2 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Topscript rt drymix dt18 plus, by Enzynomics (1 mentions)
M mlv cdna synthesis kit, by Enzynomics (1 mentions)
96 well reaction plate, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit cdna kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Nanodrop lite spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Step one plus real time pcr kit, by Thermo Fisher Scientific (1 mentions)
Stepone software, by Thermo Fisher Scientific (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
19 protocols using topreal qpcr 2 premix sybr green with low rox
1
Developmental Expression of MviPBANR-B and -C
2
Quantitative PCR Analysis of Immunomodulatory Genes
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Total RNA was isolated from cell pellets using an RNeasy mini kit (QIAGEN, Hilden, Germany), and first-strand cDNA was synthesized using RT Drymix (Enzinomics, Daejeon, Korea) according to the manufacturer’s instructions. The quantity of mRNA was determined by real-time polymerase chain reaction (PCR; Agilent Technologies, Santa Clara, CA, USA) using TOPreal™ qPCR 2× PreMIX (SYBR Green with low ROX) (Enzynomics). The following primers were used for analyses: mIL-10 (Forward: 5′-GCACTGCTATGCTGCCTGCTCTTACTGA-3′, Reverse: 5′-AGCTTCTCACCCAGGGAATTCAAATGCT-3′), mTGF-β1 (Forward: 5′-CTCCCGTGGCTTCTAGTGC-3′, Reverse: 5′-GCCTTAGTTTGGACAGGATCTG-3′), iNOS (Forward: 5′-CAAATCCTACCAAAGTGACCTGAAA-3′, Reverse: 5′-TACTGTGGACGGGTCGATGTC-3′), Arg1 (Forward: 5′-TCCACCCTGACCTATGTGTCATTT-3′, Reverse: 5′-CGTCTCGCAAGCCAATGTACA-3′). All gene expression levels were normalized using the glyceraldehyde-3-phosphate dehydrogenase gene.
3
qRT-PCR Analysis of VCP and LC3 Expression
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Total RNA was isolated using the ImProm-II Reverse Transcriptase kit (Promega, Madison, WI, USA) and cDNA was synthesized. qRT-PCR was performed with the CFX96 Real-Time System (Bio-Rad) and TOPreal™ qPCR 2× PreMIX (SYBR Green with low ROX) (Enzynomics, Daejeon, Republic of Korea). β-actin was used as the reference gene for normalization. Primers used for VCP, LC3 and β-actin were as follows: VCP forward, 5′- AGAGCAACCTTCGTAAAGCC-3′, and reverse, 5′-AACAACTGAGACACGATGCG-3′; LC3 forward 5′- AGTGGAAGATGTCCGGCTCA-3′, and reverse, 5′- GCTTCTCACCCTTGTATCGCT-3′; β-actin forward, 5′- GTCCACCCGCGAGTACAACCTT-3′, and reverse, 5′- TTGCACATGCCGGAGCCGTT-3′.
4
Analyzing miRNA Target Genes and Pathways
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The predicted target genes were retrieved for each differentially expressed miRNA using TargetScan (http://www.targetscan.org/vert_72/ ), and the overlapping predicted target genes were identified. The mRNA expression of predicted target genes of five or more miRNAs was explored using qRT-PCR [16 (link), 17 (link)]. The ViiA7 real-time PCR system (Applied Biosystems; Carlsbad, CA, USA) and TOPreal™ qPCR 2× PreMIX (SYBR Green with low ROX; Enzynomics; Daejeon, Korea) were used. The primer sets used are described in Table 1 . All primers were tested for amplification efficiency. Glyceraldehyde 3-phosphate dehydrogenase was used as a reference gene to normalize the amplicon levels using the 2–ΔΔCt method. Then, the expression fold changes of each group were calculated and compared to those of the control group. Based on the predicted target genes confirmed using qRT-PCR, the pathways were further analyzed using DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/tools.jsp ).
5
Quantitative Analysis of miRNA Target Genes
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The predicted target pathways and related genes for the dysregulated miRNAs were accessed using DIANA-mirPath v.3 (Available online: http://snf-515788.vm.okeanos.grnet.gr/ (accessed on 7 June 2021)). KEGG pathways and the related target genes were searched with a threshold of 0.8 and a false discovery rate correction [46 (link)].
The mRNA expression of the predicted target genes was analyzed using quantitative reverse-transcription-PCR (qRT-PCR) [47 (link),48 (link)]. Following reverse transcription of cDNA from purified RNA, the reagent was prepared using TOPreal™ qPCR 2× PreMIX (SYBR Green with low ROX; Enzynomics; Daejeon, Korea). PCR was conducted using the ViiA7 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). The primer sets used in this study are listed inTable 2 . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the reference gene. mRNA expression levels were quantified using the 2−ΔΔCt method.
The mRNA expression of the predicted target genes was analyzed using quantitative reverse-transcription-PCR (qRT-PCR) [47 (link),48 (link)]. Following reverse transcription of cDNA from purified RNA, the reagent was prepared using TOPreal™ qPCR 2× PreMIX (SYBR Green with low ROX; Enzynomics; Daejeon, Korea). PCR was conducted using the ViiA7 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). The primer sets used in this study are listed in
6
Rice Gene Expression in M. oryzae Infection
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Rice gene expression levels in M. oryzae-infected leaf sheath tissues were analyzed by reverse-transcription PCR (RT-PCR) and real-time quantitative PCR (qRT-PCR). Total RNAs were extracted from rice tissues using TRIzol reagent (Invitrogen), followed by first-strand cDNA syntheses using a cDNA synthesis kit (Invitrogen) according to the manufacturer’s instructions. Equal amounts of cDNAs were used as templates for RT-PCR and real-time qRT-PCR. The expression of OsNLP2, OsPBZ1, OsWRKY104, OsRbohB, OsPIP-3A, and OsWRKY90 was analyzed with gene-specific primer sets by real-time qRT-PCR using TOPreal qPCR 2× PreMIX (SYBR Green with low ROX, Enzynomics). Relative expression levels of the tested rice genes were determined by normalizing them with respect to the expression levels of rice OsUbiquitin, 18S rRNA, and OsActin as internal controls. The relative expression values were calculated using the ΔΔCt method [33 (link)]. The data are means ± SD of relative gene expression levels in leaf sheaths from three independent experiments. The list of gene-specific primers used in this study is provided in Table S1 .
7
Quantitative RT-PCR Analysis of Cochlear Gene Expression
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Quantitative RT-PCR was conducted as previously described [37 (link)]. In brief, total RNA was extracted from cochleae using TRI Reagent® (Sigma-Aldrich, St. Louis, MO, USA). TOPscriptTM RT DryMIX (dT18 plus; Enzynomics Co. Ltd., Daejeon, South Korea) was used for the reverse transcription reaction. Quantitative RT-PCR was performed with TOPreal™ qPCR 2 × PreMIX (SYBR Green with low ROX; Enzynomics, Daejeon, Korea) and oligonucleotide primers (Table 1 ) in a CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). mRNA expression levels were estimated based on the expression level of the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
8
RNA Extraction and qRT-PCR Analysis
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Invitrogen TRIzol reagent was used for RNA extraction. On the day of RNA extraction, sample cells were harvested and treated with 200 μL of the TRIzol reagent. RNA extraction was performed through instructions from manufacturers. 1 μg (HEK293T) or 200 ng (UC-MSC) of isolated total RNAs were converted to cDNA in 20-μL reaction volumes by using an M-MLV cDNA synthesis kit from Enzynomics, according to the manufacturer’s instructions. For quantitative RT-PCR (qRT-PCR), the cDNA samples were diluted five times, and 3 μL of the diluted cDNA (30 ng or 6 ng) was put in each well of a 96-well reaction plate (Applied Biosystems). TOPreal qPCR 2× PreMIX (SYBR green with low ROX, Enzynomics) served as fluorescence signals to detect target cDNA amounts. Relative mRNA expressions were determined by ΔΔCt method, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as endogenous control. Reactions were run on a StepOnePlus real-time PCR system (Applied Biosystems). All primers for qRT-PCR were listed in Table S1 .
9
RNA Extraction and RT-qPCR Analysis
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Extracting the total RNA from all isolated tissues was done by using Trizol (Invitrogen; Thermo Fisher Scientific, Inc.). Quantifying RNA concentrations was done through a Nanodrop using the NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies; Wilmington, Delaware, United States). Synthesis of cDNA was by using a High-Capacity cDNA Reverse Transcription Kit cDNA Kit (Applied Biosystems™, USA). All primers are prepared following the manufacturer’s instructions. Real-time RT-PCR was accomplished in the M × 3005P Real-Time PCR System (Agilent Stratagene, USA) by means of TOPreal™ qPCR 2 × PreMIX (SYBR Green with low ROX) (Cat.#P725or P750) (Enzynomics, Korea) following manufacturer's directions. Expression levels of the investigated assayed genes were normalized via mRNA expression of B-actin (identified housekeeping gene). The outcomes were expressed as fold-changes compared with the control group according to the 2-ΔΔCT method (Livak and Schmittgen, 2001 (link)). The primer sequences that have been used are listed in Table 1 .
10
Investigating YY1 Inhibition in Prostate Cells
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To identify the role of Yin Yang 1 (YY1) in prostate cell lines, cells were treated with 10 nM NP-001 (Adooq Bioscience, Irvine, CA, USA), a YY1 inhibitor, for 24 h. Total RNA was then extracted from each treatment and control population using Trizol reagent (Invitrogen) according to the manufacturer's instructions. RNA concentrations were measured with a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA (1 µg) was synthesized from total RNA using a First Strand cDNA Synthesis Kit (Thermo Fisher Scientific), as described by the manufacturer. Real-time PCR was performed using TOPreal™ qPCR 2× PreMIX (SYBR Green with low ROX) from Enzynomics (Daejeon, South Korea). All PCR reactions were carried out in triplicate using the StepOnePlus Real-Time PCR kit (Applied Biosystems, Carlsbad, CA, USA). The PCR primers and conditions are shown in Table S1 . mRNA levels were calculated by the 2ΔΔct method using StepOne software (Applied Biosystems). Results were compared using the Mann Whitney test and are presented as mean ± standard deviation (SD); p<0.05 was considered statistically significant. All statistical analyses were conducted using SPSS software (SPSS Statistics, Chicago, IL, USA).
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