Accupower cyclescript rt premix

Manufactured by Bioneer

Sourced in Cameroon, United States

AccuPower® CycleScript RT PreMix is a ready-to-use solution for reverse transcription. It contains all the necessary components for cDNA synthesis from RNA templates.

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Lab products found in correlation

84 protocols using accupower cyclescript rt premix

Transcriptional Analysis of Animal Cells

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Dulbecco’s Modified Eagle Medium-F12 (DMEM-F12), fetal bovine serum (FBS),
horse serum (HS), and antibiotic-antimycotic (AA) were purchased from Gibco
(Thermo Fisher Scientific, Waltham, MA, USA). Trizol reagent
(Accuzol^TM Total RNA Extraction Reagent, Bioneer, Seoul, Korea),
diethylpyrocarbonate (DEPC) water (Bioneer), cDNA transcription kit (AccuPower
CycleScript RT PreMix, Bioneer), qPCR MasterMix (Bioneer), and
nuclease-free-water (Ambion®, Austin, TX, USA) were purchased for RNA
extraction, cDNA synthesis, and quantitative real-time polymerase chain reaction
(PCR). The experimental protocols for this research were reviewed and approved
by the Institutional Animal Care and Use Committee at the National Institute of
Animal Science (NIAS-2020-437).

Kim B., Min Y., Jeong Y., Ramani S., Lim H., Jo Y., Kim W., Choi Y, & Park S. (2023). Comparison of growth performance and related gene expression of muscle and fat from Landrace, Yorkshire, and Duroc and Woori black pigs. Journal of Animal Science and Technology, 65(1), 160-174.

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Quantitative PCR Analysis of Inflammatory Markers

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Total RNA was isolated using an easy-BLUE™ RNA extraction kit (iNtRON, Daejeon, Korea) according to the procedure described by the manufacturer. The total RNA was transformed into cDNA using AccuPower® CycleScript RT PreMix (Bioneer, Daejeon, Korea). Specific primers amplified by PCR are described in Table 2. The following PCR conditions were applied for TNF-α, IL-6, IL-1β, COX-2, iNOS, HO-1, and β-actin: 35 cycles of denaturation at 94°C for 30 s, annealing at the temperature indicated in Table 2 for 30 seconds, and extension at 72°C for 30 seconds [27 (link)-31 (link)].

Oh Y.C., Jeong Y.H., Ha J.H., Cho W.K, & Ma J.Y. (2014). Oryeongsan inhibits LPS-induced production of inflammatory mediators via blockade of the NF-kappaB, MAPK pathways and leads to HO-1 induction in macrophage cells. BMC Complementary and Alternative Medicine, 14, 242.

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RNA Isolation and RT-qPCR Analysis of RAW 264.7 Cells

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Total RNA was isolated from RAW 264.7 cells using the RNeasy Mini kit (Qiagen, Germany) using the manufacturer’s standard protocol. RNA quality and quantity were determined using a NanoPhotometer (Implen, Germany). Extracted RNA was reverse-transcribed to generate complementary DNA (cDNA) using the AccuPower CycleScript RT premix (Bioneer, South Korea) according to the manufacturer’s instructions. RT-qPCR was conducted using a StepOnePlus Real-Time PCR System (Thermo Scientific) with SYBR Green PCR Master Mix (Thermo Scientific). The RT-qPCR reaction was performed in a 96-well plate containing 10 mL of SYBR Green PCR Master Mix, 2 µL of primers (5 pmol), 2 µL of cDNA template, and 6 µL of nuclease-free water. The RT-qPCR conditions were as follows: 1 cycle of 95°C for 10 min; 40 cycles of 95°C for 15 s, 60°C for 1 min, and 72°C for 40 s. The relative gene expression was determined by using the 2^–ΔΔCT comparative method (113 (link)). All data were normalized to the expression level of the β-actin housekeeping gene.

Yoon K.N., Lee S.J., Keum G.B., Song K.Y., Park J.H., Song B.S., Yu S.Y., Cho J.H., Kim E.S., Doo H., Kwak J., Kim S., Eun J.B., Lee J.H., Kim H.B., Lee J.H, & Kim J.K. (2023). Characteristics of Lactococcus petauri GB97 lysate isolated from porcine feces and its in vitro and in vivo effects on inflammation, intestinal barrier function, and gut microbiota composition in mice. Microbiology Spectrum, 12(1), e01334-23.

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Quantitative Real-Time PCR Protocol

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Bacteria were pelleted by centrifugation at 2,500 × g for 15 min. Total RNA was isolated using the QIAGEN RNeasy Mini kit. Extracted RNA was analyzed using a nanodrop ND1000 and running on a denaturing 1.5% Tris-acetate-EDTAagarose gels (80 V for 1 h) to assess RNA concentration, quality, and integrity. The RNA was DNase treated with Promega RNase-free DNase (at 37 °C for 1 h). RNA was precipitated with 1 volume isopropanol and 0.1 volume of 3 M NaOAc (pH 4.6). The suspension was incubated on ice for 20 min and centrifuged at high speed for 30 min at 4 °C. The RNA was pellet, dried, and resuspended with RNase-free MilliQ H 2 O. According to the manufacturer's instructions, 500 ng-1 μg RNA was converted into cDNA using AccuPower CycleScript RT PreMix (Bioneer, Korea). Quantitative real-time PCR was performed in a Rotor-Gene thermal cycler (Corbett 6000, Australia) using SYBR Green method (AccuPower Green Star qPCR Master Mix, Bioneer, Korea). A total volume of 20 μl reaction containing 2 μl of cDNA, 12.5 μl SYBR Green master mix, 4.5 μl nuclease-free water, and 1 μl of each primer (5 pmol) was run according to following program: an initial activation step at 94 °C for 4 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at (indicated in Table II) for 30 s, and extension at 72 °C for 20 s.

Behrooz S.K., Lida L., Ali S., Mehdi M., Rasoul M., Elnaz O., Farid B.T, & Gholamreza I. (2018). Study of MazEF, sam, and phd-doc putative toxin-antitoxin systems in Staphylococcus epidermidis. Acta microbiologica et immunologica Hungarica, 65(1).

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Pluripotency and Lineage Marker Analysis

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Co-cultured E14-EGFPs were harvested and sorted using FACS Aria. Semi-quantitative reverse transcription (RT-PCR) for Oct4, Nanog, Sox2, Pax6, Sox1, Mixl1, T, Gata4, Sox17, Otx2, Nestin, and Gapdh was performed using standard procedures. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and 1 µg of total RNA was reverse-transcribed with an Accupower CycleScript RT Premix (Bioneer, Seoul, Korea) according to the manufacturer’s instructions. cDNA was subjected to qPCR using Powerup SYBR Green master mix (Applied Biosystems, CA, USA). The primer sequences are listed in Table 1.

Kim Y.R., Jang S.W., Han J.H., Na G.R., Jang H, & Choi H.W. (2022). The Effects of Co-Culture of Embryonic Stem Cells with Neural Stem Cells on Differentiation. Current Issues in Molecular Biology, 44(12), 6104-6116.

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RNA Extraction and Gene Expression Analysis

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The Trizol method was used to extract total RNA from HaCaT cells as per the manufacturer’s instructions (Invitrogen, Waltham, MA, USA). The extracted RNA was then subjected to reverse transcription using AccuPower® CycleScript™ RT PreMix (Bioneer, Daejeon, Republic of Korea) to produce cDNA. To assess the mRNA expression levels of the target genes, PCR analysis was performed. The results were normalized to the expression levels of the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primer sequences utilized in this analysis can be found in Supplementary Table 2.

Lee S.H., Lee N.Y., Choi S.H., Oh C.H., Won G.W., Bhatta M.P., Moon J.H., Lee C.G., Kim J.H., Park J.L, & Park J.T. (2023). Molecular mechanism of the anti-inflammatory and skin protective effects of Syzygium formosum in human skin keratinocytes. Food Science and Biotechnology, 33(3), 689-697.

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Quantification of Tight Junction Gene Expression

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1 × 10⁵ Caco2 cells per well were plated in 6-well tissue culture-treated plates. At the end of the experiment, RNA was obtained using TRIzol reagent and chloroform. Reverse transcription was conducted in a 20 μl reaction with 1 μg of total RNA transformed into cDNA using AccuPower Cycle Script RT Premix (Bioneer). The measurements of ZO-1, ZO-2, and ZO-3, claudin-1, claudin-2, and claudin-7, occludin, and β-actin mRNA were conducted under the following conditions: 45 cycles of 95°C for 10 s, 60°C for 20 s, and 72°C for 30 s using a LightCycler 480 II (Roche, Rotkreuz, SWI). The mRNA level in each sample was quantified using the cycle threshold (Ct) value. The target genes were normalized relative to the reference gene actin.

Park K.I., Kim D.G., Lee B.H, & Ma J.Y. (2016). Fermented Herbal Formulas KIOM-MA128 Ameliorate IL-6-Induced Intestinal Barrier Dysfunction in Colon Cancer Cell Line. Mediators of Inflammation, 2016, 6189590.

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Cloning and Characterization of E. granulosus EG95

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The total RNA was extracted from the oncosphere using TRIzol reagent followed by quantification using a spectrophotometer (Nano Drop) at wavelengths of 230, 260, and 280 nm. A transcription kit, the AccuPower® CycleScript RT PreMix, k-2046 (BIONEER), was applied to amplify all mRNA into complementary DNA (cDNA) according to the manufacturer’s instruction (11 (link)). The open reading frame of E. granulosus EG95 was amplified for 35 cycles (an initial denaturation at 94°C for 5 min, followed by 35 cycles of 30 s at 94°C, 30 s at 53°C, 30 s at 72°C and final extension at 72°C for 10 min). PCR amplification was performed using the following primers in which restriction sites were inserted as underlined below. F: 5′-CGGAATTCATGGCATTCCAGTTATGTCTC-3′ (EcoRI) and R: 5′-GCCTCGAGTCAAGTAAGGACAAC-3′ (XhoI) (12 ).
The PCR product was ligated into the pET28a (+) vector (Novagen) by T₄ DNA ligase (Thermo scientific) at the EcoRI and XhoI sites to construct recombinant plasmid pET28a/His-EG95. E.coli Top10 was transformed with the resultant recombinant plasmid. The insert was purified by a mini columns plasmid purification kit (GeneAll, Korea) and confirmed by restriction enzyme digestion, PCR and sequencing.

MAZAHERI N., DALIMI A., PIRESTANI M., JAMEIE F., MOHEBALI M, & ROKNI M.B. (2017). Construction and Identification of a Recombinant Plasmid Encoding Echinococcus granulosus Oncosphere Antigen (EG95). Iranian Journal of Parasitology, 12(4), 490-497.

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Validating RNA-seq Data by qRT-PCR

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qRT-PCR was performed to validate the RNA-seq data of the selected DEGs. Briefly, 1 µg of total RNA was reverse-transcribed into cDNA using AccuPower^® CycleScript RT PreMix (Bioneer, Seoul, Korea) following the manufacturer’s protocol, and aliquots were stored at –20°C. The cDNA was amplified using custom sequence-specific primers (Cosmogenetech, Seoul, Korea) and detected using SYBR^® Green (Solgent, Daejeon, Korea). The primer sequences of the selected DEGs are shown in Supplementary Table 1. The input concentration for cDNA synthesis was 2.5 µg/µL. The synthesis conditions were as follows: 94°C for 1 min (denaturing step), followed by annealing at primer-specific temperature for 1 min, and then 72°C for 45 s. Triplicate samples were done during qRT-PCR analyses. Values were normalized relative to Gapdh. The results were shown as relative expression levels calculated through the 2^−ΔΔCT method (VanGuilder et al., 2008 (link)).

Sayson L.V., Kim M., Jeon S.J., Custodio R.J., Lee H.J., Ortiz D.M., Cheong J.H, & Kim H.J. (2022). Differentially Expressed Genes in Period 2-Overexpressing Mice Striatum May Underlie Their Lower Sensitivity to Methamphetamine Addiction-Like Behavior. Biomolecules & Therapeutics, 30(3), 238-245.

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Evaluating Inflammatory Cytokine Expression in Spleen

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The mRNA expression of IL-4, IL-6, TNF-α, and IFN-γ was evaluated in spleen tissues by RT-qPCR as previously described (40 (link)). Spleen tissues stored at -80˚C were thawed on ice and homogenized in an e-tube containing 1 ml Total RNA isolation solution (RiboEX^™; GeneAll Biotechnology Co., Ltd.) using a homogenizer. Gene extraction solution (GeneAll^®Hybrid-R^™; GeneAll Biotechnology Co., Ltd.) was used to extract RNA from the tissues. The concentration of extracted RNA was measured with a NanoDrop ND-1,000 Spectrometer (NanoDrop products), and cDNA was obtained using AccuPower^® CycleScript RT PreMix (Bioneer Corporation). Samples were stored at -80˚C until further analysis. RT-qPCR was run using SYBR^® Green (SolGent Co., Ltd.). RT-qPCR was performed. The sequences of the primers used are presented in Table I. The relative expression levels were normalized to endogenous control and were expressed as 2^-ΔΔCq^{(41 (link))}.

Kim Y.L., Lim H.S, & Lee S.M. (2021). Effect of low-level laser intervention on dermatitis symptoms and cytokine changes in DNCB-induced atopy mouse model: A randomized controlled trial. Experimental and Therapeutic Medicine, 22(5), 1196.

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