Total RNA was extracted via RiboEx reagent (GeneAll, Korea) according to the manufacturer’s protocol. The total RNA of clinical tissue was extracted via grinding them in 1 ml RiboEx reagent using porcelain glass grinder. Complete DNA (cDNA) was synthesized from 1 μg of total RNA using cDNA synthesis kit (Biofact, Korea). Then, qRT-PCR technique was applied to assess gene expressions. First, total RNA of tumor tissues and cells were extracted using the RiboEx reagent (GeneAll, Korea) according to the manufacturer’s protocol. Then, total RNA was quantified and qualified by NanoDrop Spectrophotometer (Thermo Fisher Scientific, USA) and agarose gel electrophoresis, respectively. Thereafter, cDNA was synthesized by a cDNA synthesis kit (Takara, Japan). Finally, mRNA expressions were examined by the Roche LightCycler system (Roche, Germany) by applying SYBR Premix Ex Taq (Takara, Japan). Relative Nrf2, PD-L1, and CD80 mRNA expressions in tissue samples were analyzed through the 2–ΔΔCt method, in which endogenous β actin was considered as an internal control. However, for the assessment of raw data from the cell samples, Pffafl method was applied and GAPDH gene was considered as an internal control. All of the experiments were performed at least in triplicate.
Riboex reagent
Manufactured by GeneAll
Sourced in Cameroon, Portugal
RiboEx is a reagent used for the isolation and purification of total RNA from a variety of biological samples, including cells, tissues, and microorganisms. It is a single-step method that utilizes the guanidinium thiocyanate-phenol-chloroform extraction technique to effectively extract high-quality RNA.
Automatically generated - may contain errors
Lab products found in correlation
Hybrid r kit, by GeneAll (6 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (6 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (6 mentions)
Lightcycler 96, by Roche (5 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Sybr premix ex taq, by Takara Bio (4 mentions)
Realq plus 2x master mix, by Ampliqon (3 mentions)
All in one rt mastermix kit, by Applied Biological Materials (3 mentions)
Lightcycler 96 instrument, by Roche (3 mentions)
Cdna synthesis kit, by Toyobo (3 mentions)
Sybr green master mix, by Biofact (3 mentions)
Cdna synthesis kit, by Biofact (3 mentions)
Cfx connect real time system, by Bio-Rad (3 mentions)
Nanodrop, by Thermo Fisher Scientific (3 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Topreal sybr green qpcr premix, by Enzynomics (2 mentions)
Revertra acetm qpcr rt kit, by Toyobo (2 mentions)
Sybr green pcr master mix, by Bio-Rad (2 mentions)
81 protocols using riboex reagent
1
Quantitative Assessment of Nrf2, PD-L1, and CD80 Expressions
2
Total RNA Extraction and Real-Time PCR Analysis
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Cells and tissues were homogenized using RiboEXTM reagent (GeneAll; Seoul, Korea), chloroform was added, and the solution was shaken vigorously for 15 min. The aqueous phase was then transferred to fresh tubes, isopropanol was added, and the solution was incubated for 15 min at 4 °C and centrifuged for 15 min at 12,000× g. The supernatants were removed, and the pellets were washed with 75% ethanol and centrifuged for 5 min at 8000× g. The RNA pellets obtained were dried and dissolved in diethylpyrocarbonate water, and the mRNA concentrations were calculated. mRNA was reverse transcribed to cDNA using SuPrimeCript RT Premix (Genetbio Inc.; Daejeon, Korea). Real-time PCR analysis was performed using SYBR green master mix (BIOLINE, London, UK) and the CFX Connect System (Bio-Rad Inc.).
3
RNA Extraction and Real-Time PCR
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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.
4
Analyzing Adipogenesis Genes in 3T3-L1 Cells
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The expression of various genes associated with lipogenesis and adipogenesis in
3T3-L1 cells was analyzed by qRT–PCR. Total RNA was extracted using the
RiboExTM reagent (GeneAll Biotechnology, Seoul, Korea). The RNA was
reverse-transcribed into cDNA using the ReverTra AceTM qPCR RT kit (Toyobo,
Japan). qRT–PCR was carried out using the SYBR Green PCR Master Mix on
the CFX Connect Real-Time PCR system (both from Bio-Rad Laboratories). The mRNA
levels of 36B4 were used as an internal control. The specific primer sequences
are listed inTable 1 .
3T3-L1 cells was analyzed by qRT–PCR. Total RNA was extracted using the
RiboExTM reagent (GeneAll Biotechnology, Seoul, Korea). The RNA was
reverse-transcribed into cDNA using the ReverTra AceTM qPCR RT kit (Toyobo,
Japan). qRT–PCR was carried out using the SYBR Green PCR Master Mix on
the CFX Connect Real-Time PCR system (both from Bio-Rad Laboratories). The mRNA
levels of 36B4 were used as an internal control. The specific primer sequences
are listed in
5
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)].
6
Quantifying LINC00968 Expression in Tissues and Cell Lines
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The total RNA was extracted from tissue samples and cell lines using RiboExTM reagent (GeneAll, Korea). The extracted RNAs were treated with DNase I (EN0521, Thermo Fisher scientific, United States). Then, reverse transcription into cDNA was performed by a 5X All-In-One RT MasterMix kit (Applied Biological Materials, CA). Real-time PCR was performed in duplicate using RealQ Plus 2x master mix (AMPLIQON, Denmark) in LightCycler® 96 instrument (Roche Diagnostics, Mannheim, Germany). The β2M housekeeping gene was used as a normalizer. The primer sequences of LINC00968 were 5′- CCAGACTCCTCAGCCTGAAAT-3′ (forward) and 5′-GTCCCAAACGCAGACCATTTT-3′ (reverse). Also, the primer sequences of β2M were 5′- AGATGAGTATGCCTGCCGTG-3′ (forward) and 5′- GCGGCATCTTCAAACCTCCA-3′ (reverse). The relative expression was calculated using the 2-ΔΔCT method.
7
qRT-PCR Workflow for Gene Expression
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Total RNA was extracted with the use of the RiboExTM reagent (GeneAll Biotechnology, Korea). The synthesis of cDNA was performed by using the ReverTra AceTM qPCR RT kit (Toyobo, Japan). Quantitative real-time PCR was performed by using a CFX Connect Real-Time PCR system (Bio-Rad Laboratories) and SYBRGreen PCR Master Mix (Bio-Rad Laboratories). 36B4 was used as an internal control in order to calculate the expression levels. The sequences of the primers used in the experiment are the same as in a previous study (Yeon et al., 2021 (link)).
8
Grass Puffer Brain and Pituitary Total RNA Extraction
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RiboExTM reagent (GeneAll, Seoul, Korea) were added to the brains and
pituitary tissues collected from grass puffer, and the samples were completely
homogenized using a homogenizer. Total RNA treated with RQ NRase-Free DNase
(Promega, Madison, USA). Only the DNase-treated total RNA with 1.7-2.1 ratio of
A260/A280 nm was used in the experiment using Nano Vue (GE Healthcare,
Ver.1.0.1, London, UK). cDNA was synthesized using PrimeScript 1st strand cDNA
synthesis Kit (Takara, Japan) with the template of DNase-treated total RNA 0.5
μg.
pituitary tissues collected from grass puffer, and the samples were completely
homogenized using a homogenizer. Total RNA treated with RQ NRase-Free DNase
(Promega, Madison, USA). Only the DNase-treated total RNA with 1.7-2.1 ratio of
A260/A280 nm was used in the experiment using Nano Vue (GE Healthcare,
Ver.1.0.1, London, UK). cDNA was synthesized using PrimeScript 1st strand cDNA
synthesis Kit (Takara, Japan) with the template of DNase-treated total RNA 0.5
μg.
9
Expression Analysis of lncRNAs in Tumor Samples
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Total RNA was extracted from tumor and adjacent non-tumor tissue samples and also cell lines, using RiboEx TM reagent (GeneAll). The extracted RNAs were treated with DNase I (EN0521, Thermo Fisher scienti c, United States). Then, one µg RNA was reverse transcribed into cDNA by 5X All-In-One RT MasterMix kit (Applied Biological Materials). Real time PCR was performed in duplicate, using RealQ Plus 2x master mix (AMPLIQON) in Roche LightCycler ® 96 instrument. The β2M housekeeping gene was used as normalizer. The primer sequences of FOXD2-AS1 were 5′-CTGTTCTCGGCTCTGGGAAAG-3′ (forward) and 5′-GTGCAATCGTTCCGCTGTG-3′ (reverse) and the primer sequences of LINC00968 were 5′-CCAGACTCCTCAGCCTGAAAT-3′ (forward) and 5′-GTCCCAAACGCAGACCATTTT-3′ (reverse). The relative expression was calculated using 2 -∆∆CT method.
10
Quantifying LINC00968 Expression in Tumor Samples
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Total RNA was extracted from tumor and adjacent non-tumor tissue samples and cell lines as well, using RiboEx TM reagent (GeneAll, Korea). The extracted RNAs were treated with DNase I (EN0521, Thermo Fisher scienti c, United States). Then, reverse transcription into cDNA was performed by 5X All-In-One RT MasterMix kit (Applied Biological Materials, CA). Real time PCR was performed in duplicate, using RealQ Plus 2x master mix (AMPLIQON, Denmark) in LightCycler ® 96 instrument (Roche Diagnostics, Mannheim, Germany). The β2M housekeeping gene was used as normalizer. The primer sequences of LINC00968 were 5′-CCAGACTCCTCAGCCTGAAAT-3′ (forward) and 5′-GTCCCAAACGCAGACCATTTT-3′ (reverse). Also, the primer sequences of β2M were 5′-AGATGAGTATGCCTGCCGTG-3′ (forward) and 5′-GCGGCATCTTCAAACCTCCA-3′ (reverse). The relative expression was calculated using 2 -∆∆CT method.
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