Powerup sybr green

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

PowerUp SYBR Green is a real-time PCR reagent designed for quantitative gene expression analysis. It contains a proprietary dye that binds to double-stranded DNA and emits a fluorescent signal during the amplification process, enabling real-time monitoring of the PCR reaction.

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182 protocols using powerup sybr green

Quantitative Real-Time RT-PCR Analysis of Gene Expression

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Total RNA was isolated from cells using the TRIzol reagent (Invitrogen). Next, 1 μg total RNA was reverse transcribed into cDNA using the GoScript™ reverse transcription system (Promega, USA) according to the manufacturer’s instructions. Analyses were performed utilizing PowerUp™ SYBR™ Green on a ABI QuantStudio 5 real-time fluorescence detection PCR system. The expression levels of specific genes are reported as ratios to GAPDH expression in the same master reaction. The primers used for real-time RT-PCR are listed in the Supplementary Table 1.

Wang Y., Yi K., Liu X., Tan Y., Jin W., Li Y., Zhou J., Wang H, & Kang C. (2021). HOTAIR Up-Regulation Activates NF-κB to Induce Immunoescape in Gliomas. Frontiers in Immunology, 12, 785463.

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Quantitative PCR Analysis of Gene Expression

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RNA was isolated from cell pellets using RNEasy Plus isolation kits (Qiagen). RNA quality and concentration were measured on a NanoDrop spectrophotometer and 0.05μg/μL was used for cDNA synthesis reactions with random hexamers and Taqman Multiscribe Reverse Transcriptase. PowerUp Sybr Green (ABI), 12μM cDNA, and primers (Supp. Table 1) were used for qPCR assays on a QuantStudio 12k Flex System (ThermoFisher). qPCR data was expressed relative to 18s RNA, and fold changes between 1,25D treated and control samples were calculated as ΔΔC_T. The data shown represent the average fold change of three cell passages (biological replicates) +/− SD.

DeSantis K.A., Robilotto S.L., Matson M., Kotb N.M., Lapierre C.M., Minhas Z., Leder A.A., Abdul K., Facteau E.M, & Welsh J. (2020). VDR in Salivary Gland Homeostasis and Cancer. The Journal of steroid biochemistry and molecular biology, 199, 105600.

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Quantitative PCR Analysis of Gene Expression

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RNA was isolated from human C4–2 cells using Trizol reagent (Invitrogen) according to the manufacturer’s instructions followed by purification on an RNeasy column (Qiagen). First-strand cDNA synthesis was performed using Superscript II (Invitrogen) followed by quantitative PCR using PowerUp SYBR Green according to manufacturer’s instructions. KiCqStart SYBR Green Primers were obtained from Sigma-Aldrich (Table S7). mRNA expression was normalized to house keeper (UBC) gene expression, the expression of which was unaltered between conditions.

Penfold L., Woods A., Pollard A.E., Arizanova J., Pascual-Navarro E., Muckett P.J., Dore M.H., Montoya A., Whilding C., Fets L., Mokochinski J., Constantin T.A., Varela-Carver A., Leach D.A., Bevan C.L., Nikitin A.Y., Hall Z, & Carling D. (2023). AMPK activation protects against prostate cancer by inducing a catabolic cellular state. Cell reports, 42(4), 112396.

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Quantitative PCR Analysis of Gene Expression

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Cytoplasmic and Nuclear RNA Isolation

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Total RNA was extracted from EC cells using Trizol reagent (Invitrogen, CA, USA). The Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek Corp., B.C, CAN) was used to isolate cytoplasmic and nuclear RNA fragments. Then, reverse transcription was conducted using SuperScript™ reverse transcriptase (Invitrogen) and gene amplification and quantification were tested by PowerUp™ SYBR™ Green (Invitrogen) with the QuantStudio™ 7 Pro system (Invitrogen). GAPDH was used as the reference control for quantifying SLERT and BDNF expression.

Tian J., Cheng H., Wang N, & Wang C. (2023). SLERT, as a novel biomarker, orchestrates endometrial cancer metastasis via regulation of BDNF/TRKB signaling. World Journal of Surgical Oncology, 21, 27.

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qPCR Protocol for Gene Expression

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cDNA was generated from 1 μg of RNA using the SuperScript IV first-strand synthesis system (Invitrogen). qPCR was performed in 10- or 20-μl reactions using 2 or 5 ng of cDNA as the template and 0.4 μM concentrations of the appropriate primer pairs (see Data Set 2B). Each sample was analyzed in technical duplicates. Amplicons were detected with PowerUP SYBR green (Invitrogen) in a QuantStudio 6 Flex real-time PCR system (Applied Biosystems) or a CFXConnect Real-Time PCR Instrument (Bio-Rad Laboratories). C_q values were calculated using LinRegPCR after correcting for amplicon efficiency. C_q values of technical duplicates were typically within ±0.25 of each other.holB and tufA, which encode DNA polymerase III and elongation factor Tu, respectively, were used as reference genes (see Data Set S2B). Their transcription levels remained constant in all of the experimental conditions tested here. holB was used as the reference gene in all the data presented here because its C_q values were closer to the dynamic ranges of cop genes, adcAII, cadD, and siaA, but the results were identical with when tufA was used as the reference.

Stewart L.J., Ong C.L., Zhang M.M., Brouwer S., McIntyre L., Davies M.R., Walker M.J., McEwan A.G., Waldron K.J, & Djoko K.Y. (2020). Role of Glutathione in Buffering Excess Intracellular Copper in Streptococcus pyogenes. mBio, 11(6), e02804-20.

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Age-Synchronized Worm RNA Extraction

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For experiments using whole worms, at least 500 age-synchronized worms in biological triplicate for each condition were harvested, washed three times with M9, and resuspended in 500 μl of TRIzol. To extract total RNA, worm or tissue pellets in TRIzol underwent six freeze-thaw cycles in a dry ice-ethanol bath. RNA was extracted according to the TRIzol procedure and resuspended in ribonuclease (RNase)– and deoxyribonuclease-free water. RNA from whole worms was quantified using the Qubit 2.0 fluorometer (Invitrogen), and at least 500 ng of total RNA per condition was used for cDNA synthesis (29 (link)). Total RNA was then reverse-transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche), based on the manufacturer’s instructions. RT-qPCR was performed using diluted cDNA on a Quantsudio3 (Applied Biosystems) with PowerUp SYBR green (Invitrogen). Primers used in RT-qPCR are listed in table S2. Melt curves were examined to ensure primer specificity. Results were analyzed using the standard ΔΔC_T method, and values were normalized to rpl-32 or act-1 as an internal control. All data shown represent at least three biologically independent samples.

Hamsanathan S., Anthonymuthu T., Han S., Shinglot H., Siefken E., Sims A., Sen P., Pepper H.L., Snyder N.W., Bayir H., Kagan V, & Gurkar A.U. (2022). Integrated -omics approach reveals persistent DNA damage rewires lipid metabolism and histone hyperacetylation via MYS-1/Tip60. Science Advances, 8(7), eabl6083.

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Quantitative RT-PCR Transcriptional Analysis

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RNA was extracted using RNeasy Micro or Mini kit (Qiagen) according to the manufacturer’s protocol, and complementary DNA (cDNA) was synthesized from extracted RNA using EVO Script Universal cDNA Master (#07912455001; Roche). PowerUp Sybr Green (#A25742; Invitrogen) was used for quantitative reverse transcription polymerase chain reaction (PCR) and was performed on the QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific). Primers used are listed in supplemental Table 1.
Analysis was performed using the comparative threshold cycle method (2^−ΔΔCt). Gene expression levels were normalized to housekeeping genes (Actin).

Rethnam M., Tan D.Q., Tan S.H., Li J., Yokomori R., Li Y., Yang H., Sanda T, & Suda T. (2022). Loss of METTL3 attenuates blastic plasmacytoid dendritic cell neoplasm response to PRMT5 inhibition via IFN signaling. Blood Advances, 6(18), 5330-5344.

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Measuring Gene Expression in C. elegans

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For experiments using whole worms, at least 500 age-synchronized worms in biological triplicate for each condition were harvested, washed three times with M9 and resuspended in 500 μl Trizol. To extract total RNA, worm or tissue pellets in Trizol underwent six freeze-thaw cycles in a dry ice-ethanol bath. RNA was extracted according to the Trizol procedure, and resuspended in RNase-and DNase-free water. RNA from whole worms was quantified using the Qubit 2.0 fluorometer (Invitrogen), and at least 500 ng total RNA per condition was used for cDNA synthesis 37 (link) . Total RNA was then reverse-transcribed using Transcriptor First Strand cDNA Synthesis Kit (Roche), based on the manufacturer's instructions. RT-qPCR was performed using diluted cDNA on a Quantsudio3 (applied biosystems) with PowerUp SYBR green (Invitrogen). Primers used in RT-qPCR are listed in supplementary table 2. Melt curves were examined to ensure primer specificity. Results were analyzed using the standard ΔΔC T method and values were normalized to rpl-32 or act-1 as an internal control. All data shown represent at least three biologically independent samples.

Hamsanathan S., Anthonymuthu T.S., Mullett S.J., Wendell S.G., Han S., Shinglot H., Siefken E., Sims A., Sen P., Pepper H.L., Snyder N.W., Bayır H., Kagan V.E., & Gurkar A.U. (2021). Persistent DNA damage rewires lipid metabolism and promotes histone hyperacetylation via MYS-1/Tip60.

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Optimized RNA Extraction and qPCR Analysis

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Cells were seeded at 500,000 cells/well in a 6-well plate. RNA was collected from GDNF modulated cells 24 h later. EGR1-modulated cells were treated with +/- DOX for 24 h prior to RNA collection. For all cell lines, TRI Reagent (Molecular Research Center, Inc., cat #TR118) was used to extract RNA following the manufacturer’s instructions. cDNA was made using High-Capacity RNA-to-cDNA kit (Applied Biosystems, cat #4387406), in accordance with the manufacturer’s instructions, and then was diluted 1:5 in molecular grade water. PowerUp SYBR Green was used according to the manufacturer’s instructions (Life Technologies cat #A25741). ∆∆Ct method was used for all qPCR analyses. Data are represented as mean ±SEM (n≥3) unless otherwise mentioned. qPCR primers are found in Table S1.

Marks B.A., Pipia I.M., Mukai C., Horibata S., Rice E.J., Danko C.G, & Coonrod S.A. (2023). GDNF-RET signaling and EGR1 form a positive feedback loop that promotes tamoxifen resistance via cyclin D1. BMC Cancer, 23, 138.

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