RNA was extracted from the cells using the RiboEXTM reagent (GeneAll, Republic of Korea) according to the manufacturer's instructions. The extracted RNA was converted to cDNA using the SmartGene compact cDNA Synthesis kit (SMART GENE, Republic of Korea). Real-time PCR was performed using the TOPrealTM SYBR Green qPCR PreMIX (Enzynomics, Republic of Korea) on a QuantStudio™ 1 Real-Time PCR system (Applied Biosystems, USA). The expression levels of the target genes were normalized to the housekeeping gene beta-actin, and the fold change was calculated using the 2-ΔΔCT method [21 (link)]. The primer sequence will be provided upon request.
Topreal sybr green qpcr premix
Manufactured by Enzynomics
Sourced in United States, Germany
TOPreal™ SYBR Green qPCR PreMIX is a ready-to-use solution for quantitative real-time PCR (qPCR) experiments. It contains SYBR Green I dye, DNA polymerase, and necessary reaction components.
Automatically generated - may contain errors
Lab products found in correlation
Quantstudio 1 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Riboex reagent, by GeneAll (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Topscript cdna synthesis kit, by Enzynomics (2 mentions)
Superiorscript 3 cdna synthesis kit, by Enzynomics (1 mentions)
Linegene 9600 plus machine, by Bioer (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rnaiso plus kit, by Takara Bio (1 mentions)
Linegene 9600 plus system, by Bioer (1 mentions)
Trizol reagent, by Qiagen (1 mentions)
Steponeplus software, by Thermo Fisher Scientific (1 mentions)
Quantstudio 1, by Thermo Fisher Scientific (1 mentions)
Mysticq microrna cdna synthesis mix, by Merck Group (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Tri reagent, by Thermo Fisher Scientific (1 mentions)
Nanodrop lite spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Hybrid r, by GeneAll (1 mentions)
16 protocols using topreal sybr green qpcr premix
1
RNA Extraction and Real-Time PCR
2
Comprehensive RNA Analysis Pipeline
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RNA extraction, complementary DNA (cDNA) synthesis, and RT-qPCR were conducted in accordance with our previous studies [76 (link)]. Briefly, cDNA was made using the SuperiorScript III cDNA synthesis kit (#EZ405S, Enzynomics, Daejeon, Republic of Korea). The cDNA was diluted with RNase-free water to a final concentration of 8 ng/µL, and the samples were kept at −80 °C. RT-qPCR was carried out using TOPrealTM SYBR Green qPCR PreMix (#RT500M, Enzynomics, Daejeon, Republic of Korea) and the LineGene 9600 Plus machine (BIOER, Hangzhou, China) following the manufacturer’s instructions. The primers for RT-qPCR are shown in Table 1 . The annealing temperature for the reaction was 58 °C, and the built-in software created the amplification curves and calculated the threshold cycle values. The GAPDH reference gene was used to normalize all the readouts. Data were reported as the mean relative values compared to the CON group using the 2−∆∆CT method.
3
Quantitative RT-PCR Analysis of B16F10 Cells
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The RNA was isolated from B16F10 melanoma cells using the RiboEXTM reagent (GeneAll, Seoul, Korea), and 1 µg of total RNA was used for cDNA synthesis by using a SmartGene Compact cDNA Synthesis Kit (SMART GENE, Daejeon, Korea). Quantitative real-time PCR was performed using the TOPrealTM SYBR Green qPCR PreMIX (Enzynomics, Daejeon, Korea) and QuantStudioTM 1 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The expression levels of target genes were calculated using the 2−ΔΔCT method [33 (link)].
4
Quantitative RT-PCR Analysis of Gene Expression
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qRT-PCR analysis was performed with reference to Song et al. (2021) and the instructions of the manufacturer of the reagent [15 (link)]. Following the manufacturer’s directions for the RNAiso Plus kit (Takara Bio, Inc.), total RNA was extracted from the colon tissue. cDNA was synthesized from total RNA using PrimeScript RT Master Mix (Takara Bio, Inc.). The TOPrealTM SYBR green qPCR Premix (Enzynomics, Daejeon, Republic of Korea) and a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) were used to carry out the qRT-PCR. The relative expression of the target gene was determined using the 2 −ΔΔCt method and normalized to that of the internal reference GAPDH.
5
Hippocampal Gene Expression Analysis
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RNA extraction, cDNA synthesis, and RT-qPCR were performed as described previously (Weerasinghe-Mudiyanselage et al., 2022b (link)). RNA extraction from hippocampal tissue (n=5/group) was conducted using TRIzol reagent according to the manufacturer’s instructions (#74106, Qiagen, Germany). Reverse transcription was performed using a Superscript™ III cDNA Synthesis Kit (#EZ405S, Enzynomics, South Korea). The resulting cDNA was diluted with RNase-free water to achieve a final concentration of 8 ng/µL, with the samples then stored at −70°C. RT-qPCR was performed using TOPrealTM SYBR Green qPCR PreMix (#RT500M, Enzynomics, South Korea) and the LineGene 9600 Plus system (BIOER, China) following the manufacturer’s instructions. All RT-qPCR primers are listed in Table 1 . The annealing temperature for the reaction was 58°C, and the built-in software generated the amplification curves and threshold cycle values. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as the reference gene to normalize all readings. Data were expressed as mean relative values compared to the values of the control group via the 2−∆∆CT method (Livak & Schmittgen, 2001 (link)).
6
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from MC 38 cells using an RNeasy mini kit (Hilden, Germany, Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using TOPscript RT DryMIX dT18plus (Daejeon, Korea, Enzynomics) according to the manufacturer’s instructions. Gene expression was measured using qRT-PCR, and expression data were handled using StepOne PlusTM software (Waltham, MA, USA, Applied Biosystems). qRT-PCR amplification was achieved using TOPrealTM SYBR Green qPCR PreMIX (Enzynomics). Primers were synthesized by Macrogen: Cdk1 (forward: 5′-AAGGTACTTACGGTGTGGTG-3′; reverse: 5′-CAGGTACTTCTTGAGGTCCA-3′), Cdc25a (forward: 5′-TGGACCTGTCTCCTACACTC-3′; reverse: 5′-GCTCAGTGAGAGCAGCTAAC-3ʹ), Cdc25c (forward: 5ʹ-CTACAGGACCTATCCCACCT-3′; reverse: 5′-CTCTCCACTGCTAAGATTCG-3′), Chek1 (forward: 5′-GGAGTAAGGAAATGCAGGAG-3′; reverse: 5′-GGAGAGTTAAGTGGGTGACA-3′), and Ccnb1 (forward: 5′-GCACCTGGCTAAGAATGTAG-3′; reverse: 5′-GAGCAAGTAAACACGGTAGG-3′). Mouse beta-actin was used as an internal control.
7
RNA Extraction and qPCR Analysis
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Total RNA was extracted using TRIzol Reagent (15596026; Invitrogen), and reverse transcription was performed using a TOPscriptTM cDNA Synthesis Kit (EZ005S; Enzynomics, Korea) and MystiCq® microRNA cDNA Synthesis Mix (MIRRT; Sigma) on a T100 thermal cycler (Bio-Rad, USA). Real-time qPCR was performed using TOPrealTM SYBR Green qPCR PreMIX (RT500S; Enzynomics) and QuantStudio 1 (Applied Biosystems, USA) as recommended by the manufacturer’s protocols. Gene expression was calculated using the ∆∆Ct method, and GAPDH and β-actin were used as controls. All reactions were performed in triplicate. The primer sequences used for qPCR are listed in Table 2 .
8
Zebrafish Embryo RNA Extraction and qPCR
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Twenty-five embryos from each exposure group were collected in a 1.5 mL tube at the 28-or 72 hpf stage, and then 500 µL of TRI Reagent™ solution was added (AM9738; Thermo Fisher Scientific, Waltham, MA, USA). Thereafter, 100 µL of chloroform was added to separate the organic and inorganic phases, followed by 250 µL of isopropanol to precipitate the RNA. The total RNA was extracted using the Qiagen RNeasy Mini Kit (74104, 40724; Qiagen, Hilden, Germany), and RNA concentration was measured using a Nanodrop Lite Spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized using Superscript III Reverse Transcriptase (18080093; Thermo Fisher Scientific) according to the manufacturer's instructions. Quantitative PCR (qPCR) was performed on the QuantStudio 1 Real-Time PCR system (Thermo Fisher Scientific) using SYBR Green with low ROX (RT500M; TOPreal™ SYBR Green qPCR PreMIX; Enzynomics, Daejeon, Republic of Korea); b-actin was used as the control. Each sample was run in triplicate. Gene expression was normalized to the expression level of b-actin (actb1). The transcripts were quantified using the 2 -∆∆Ct method. The qPCR primers are listed in the Supplementary file (Table S1).
9
Quantitative RT-PCR from Pancreatic Tissue
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Total RNA was isolated from pancreatic tissue or cultured cells using Hybrid-R (GeneAll), and portions (1.6 μg) of total RNA were subjected to reverse transcription using a TOPscript cDNA Synthesis Kit (Enzynomics). qRT-PCR was performed on a CFX Connect (Bio-rad) using TOPreal SYBR Green qPCR PreMIX (Enzynomics). Either 18S rRNA or TBP was used as endogenous control. The sequences of qPCR primers are listed in Tables S3 and S4 .
10
Quantification of Gene Expression in Cell Lines
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H1299 cells were treated with HAT (50–100 μg/mL) for 24 h. HUVECs were treated with HAT (25–50 μg/mL) for 24 h. Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, San Jose, CA, USA) and was quantified using a microplate reader (Molecular Devices). First-strand cDNA was synthesized using CellScript™ cDNA Master Mix (CellSafe, Suwon, Republic of Korea). The cDNA templates were diluted 10-fold in nuclease-free water and subjected to qPCR analysis using TOPreal™ SYBR Green qPCR PreMIX (Enzynomics, Daejeon, Republic of Korea) on a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The specific primer sequences and annealing temperatures for each target gene are shown in Table 2 .
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