Genelute mammalian total rna purification miniprep kit

Manufactured by Merck Group

Sourced in Belgium, United States

The GenElute Mammalian Total RNA Purification Miniprep Kit is a laboratory equipment product designed for the efficient extraction and purification of total RNA from mammalian cells and tissues. The kit utilizes a silica-based membrane technology to capture and purify the RNA, which can then be eluted for downstream applications.

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Lab products found in correlation

Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (14 mentions) Nanodrop spectrophotometer, by Thermo Fisher Scientific (6 mentions) Primescript rt master mix, by Takara Bio (4 mentions) Custom primers, by Merck Group (3 mentions) Viia 7 system, by Thermo Fisher Scientific (3 mentions) Qpcrbio sygreen mix lo rox, by PCR Biosystems (3 mentions) On column dnase 1 digestion set, by Merck Group (3 mentions) Inflammatory cytokines and receptors pathway, by Merck Group (2 mentions) Power sybr green master mix, by Thermo Fisher Scientific (1 mentions) Abi prism 7500, by Thermo Fisher Scientific (1 mentions) Rnaprotect tissue reagent, by Qiagen (1 mentions) β mercaptoethanol, by Merck Group (1 mentions) Genelute pcr clean up kit, by Merck Group (1 mentions) Iscript cdna synthesis kit, by Bio-Rad (1 mentions)

10 protocols using genelute mammalian total rna purification miniprep kit

Quantitative Real-Time PCR Protocol

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Total RNA was isolated from cells using GenElute mammalian total RNA purification mini prep kit (Sigma-Aldrich, Inc) according to manufacturer’s instructions. Total RNA concentration and quality were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Complementary cDNA was obtained using PrimeScript RT Master Mix (Takara Bio Inc, Shiga, Japan) starting from 500 ng of total RNA. cDNA was amplified by real-time quantitative polymerase chain reaction (PCR) using the Power SYBR^® Green Master Mix™ (Life Technologies, Foster City, CA, USA) and specific forward and reverse pre-designed assays (Sigma-Aldrich, Inc). The selected genes grouped by functional pathway are listed in Table 1.
PCR reactions were performed in 20 µL of final volume using the ABI PRISM 7500 (Applied Biosystems, Foster City, CA, USA). Each reaction contained 10 µL of 2× Power SYBR Green Master Mix, 400 nM concentration of each primer and 300 nM of cDNA.
After an initial denaturing step at 95°C for 10 min, the amplification proceeded with 40 cycles of a two-step profile of 15 s at 95°C and 60 s at 60°C. As final step, a melt curve dissociation analysis was performed. All experiments included non-template controls in order to check for the presence of contamination.

Palmieri A., Scapoli L., Iapichino A., Mercolini L., Mandrone M., Poli F., Giannì A.B., Baserga C, & Martinelli M. (2019). Berberine and Tinospora cordifolia exert a potential anticancer effect on colon cancer cells by acting on specific pathways. International Journal of Immunopathology and Pharmacology, 33, 2058738419855567.

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RNA Extraction from Tissue Samples

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RNA samples were collected in a mixture of RNAprotect Tissue Reagent (Qiagen, Hilden, Germany) and phosphate-buffered saline (5:1 v/v) to protect the RNA. The GenElute Mammalian Total RNA Purification Miniprep kit (Sigma-Aldrich, Bornem, Belgium) was used to extract total RNA from all samples in accordance with the manufacturer’s instructions. The extracted RNA was quantified using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, NC, USA).

Neuckermans J., Lequeue S., Claes P., Heymans A., Hughes J.H., Colemonts-Vroninks H., Marcélis L., Casimir G., Goyens P., Martens G.A., Gallagher J.A., Vanhaecke T., Bou-Gharios G, & De Kock J. (2023). Hereditary Tyrosinemia Type 1 Mice under Continuous Nitisinone Treatment Display Remnants of an Uncorrected Liver Disease Phenotype. Genes, 14(3), 693.

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RNA Extraction from Cell Cultures

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After exposure for 24 h, the cell cultures were lysed using RNA-lysis buffer containing 1% v/v β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) and stored in RNAse-free tubes at −80 °C. Total RNA was extracted and purified using the GenElute Mammalian Total RNA Purification Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA). RNA quantification was performed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Boeckmans J., Gatzios A., Heymans A., Rombaut M., Rogiers V., De Kock J., Vanhaecke T, & Rodrigues R.M. (2022). Transcriptomics Reveals Discordant Lipid Metabolism Effects between In Vitro Models Exposed to Elafibranor and Liver Samples of NAFLD Patients after Bariatric Surgery. Cells, 11(5), 893.

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Quantitative Analysis of Inflammatory Cytokines

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Total RNA was isolated from cell lines using GenElute mammalian total RNA purification miniprep kit (Sigma-Aldrich) according to manufacturer’s instructions. Pure RNA was quantified at NanoDrop 2000 spectrophotometer (Thermo Scientific).
cDNA synthesis was performed starting from 500 ng of total RNA, using PrimeScript RT Master Mix (Takara Bio Inc.). The reaction was incubated at 37°C for 15 min and inactivated by heating at 70°C for 10 s. cDNA was amplified by real-time quantitative PCR, using the ViiA^™ 7 System (Applied Biosystems).
All PCR reactions were performed in a 20 µL volume. Each reaction contained 10 µL of 2× qPCRBIO SYGreen Mix Lo-ROX (Pcrbiosystems), 400 nM concentration of each primer, and cDNA.
Custom primers belonging to the “Inflammatory Cytokines and Receptors” pathway were purchased from Sigma Aldrich. All experiments were performed including non-template controls to exclude reagents contamination. PCR was performed including two analytical replicates.
The amplification profile was initiated by 10 min incubation at 95°C, followed by two-step amplification of 15 s at 95°C and 60 s at 60°C for 40 cycles. As a final step, a melt curve dissociation analysis was performed.

Candotto V., Scapoli L., Gaudio R.M., Gianni A.B., Bolzoni A., Racco P., Lauritano D, & Cura F. (2019). Gabapentin affects the expression of inflammatory mediators on healthy gingival cells. International Journal of Immunopathology and Pharmacology, 33, 2058738419827765.

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Inflammation-related Gene Expression Analysis

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Total RNA was isolated from cell lines using GenElute Mammalian Total RNA Purification Miniprep Kit (Sigma-Aldrich, Inc,), according to manufacturer’s instructions. Pure RNA was quantified at NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).
Complementary DNA (cDNA) synthesis was performed starting from 500 ng of total RNA, using PrimeScript RT Master Mix (Takara Bio, Inc., Kusatsu, Japan). The reaction was incubated at 37°C for 15 min and inactivated by heating at 70°C for 10 s. cDNA was amplified by real-time quantitative polymerase chain reaction (PCR) using the ViiA™ 7 System (Applied Biosystems, Foster City, CA, USA).
All PCR reactions were performed in a 20 µL volume. Each reaction contained 10 µL of 2× qPCRBIO SYGreen Mix Lo-ROX (PCR Biosystems, Ltd, London, UK), 400 nM concentration of each primer, and cDNA.
Custom primers belonging to the “Inflammatory Cytokines and Receptors” pathway were purchased from Sigma-Aldrich, Inc. All experiments were performed including non-template controls to exclude reagents contamination. PCR was performed including two analytical replicates.
The amplification profile was initiated by 10 min incubation at 95°C, followed by two-step amplification of 15 s at 95°C and 60 s at 60°C for 40 cycles. As final step, a melt curve dissociation analysis, was performed.

Lauritano D., Martinelli M., Baj A., Beltramini G., Candotto V., Ruggiero F, & Palmieri A. (2019). Drug-induced gingival hyperplasia: An in vitro study using amlodipine and human gingival fibroblasts. International Journal of Immunopathology and Pharmacology, 33, 2058738419827746.

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RNA Extraction and cDNA Synthesis

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Total RNA was extracted from all samples using the GenElute Mammalian Total RNA Purification Miniprep Kit (Sigma-Aldrich) according to the manufacturer’s instructions. The isolated RNA was quantified at 260 nm using a Nanodrop spectrophotometer (Thermo Scientific, Merelbeke, Belgium). Total RNA was reverse transcribed into cDNA using iScript^TM cDNA Synthesis Kit (Bio-Rad) followed by cDNA purification with the Genelute PCR clean up kit (Sigma-Aldrich).

Colemonts-Vroninks H., Neuckermans J., Marcelis L., Claes P., Branson S., Casimir G., Goyens P., Martens G.A., Vanhaecke T, & De Kock J. (2020). Oxidative Stress, Glutathione Metabolism, and Liver Regeneration Pathways Are Activated in Hereditary Tyrosinemia Type 1 Mice upon Short-Term Nitisinone Discontinuation. Genes, 12(1), 3.

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Quantitative Real-Time PCR Analysis of Inflammatory Cytokines

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Total RNA was isolated from cell lines using GenElute mammalian total RNA purification Miniprep Kit (Sigma-Aldrich) according to the manufacturer’s instructions. Pure RNA was quantified at NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA).
Complementary DNA (cDNA) synthesis was performed starting from 500 ng of total RNA, using PrimeScript RT Master Mix (Takara Bio Inc., Kusatsu, Japan). The reaction was incubated at 37°C for 15 min and inactivated by heating at 70°C for 10 s. cDNA was amplified by real-time quantitative polymerase chain reaction (PCR) using the ViiA™ 7 System (Applied Biosystems, Foster City, CA, USA).
All PCR reactions were performed in a 20-µL volume. Each reaction contained 10 µL of 2× qPCRBIO SYGreen Mix Lo-ROX (PCR Biosystems, London, UK), 400 nM concentration of each primer, and cDNA.
Custom primers belonging to the “Inflammatory Cytokines and Receptors” pathway were purchased from Sigma-Aldrich. All experiments were performed including non-template controls to exclude reagents contamination. PCR was performed including two analytical replicates.
The amplification profile was initiated by 10 min incubation at 95°C, followed by two-step amplification of 15 s at 95°C and 60 s at 60°C for 40 cycles. As a final step, a melt-curve dissociation analysis was performed.

Candotto V., Pezzetti F., Baj A., Beltramini G., Lauritano D., Di Girolamo M, & Cura F. (2019). Phenytoin and gingival mucosa: A molecular investigation. International Journal of Immunopathology and Pharmacology, 33, 2058738419828259.

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RT-qPCR Analysis of DUBCA-hCx43 Cells

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DUBCA-hCx43 cells were seeded in a 6-well plate (15,625 cells/cm²) and exposed to the drugs for 24 h (37 °C, 5% CO²). Total RNA was extracted using a GenEluteTM Mammalian Total RNA purification Miniprep Kit (RTN70-1KT, Sigma-Aldrich, St. Louis, MO, USA) and the On-column DNase I digestion Set (DNASE70, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Isolated RNA was spectrophotometrically measured using a NanoDrop^® 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) to assess purity and quantity. A cut-off ratio between 1.8 and 2.1 for the absorption at 260/280 nm was used for assessing purity. Synthesis and amplification of cDNA, as well as the RT-qPCR analysis, were performed as explained elsewhere [84 (link)], with the exception that only 1 µg of total RNA was used to synthesize the cDNA instead of 2 µg. TaqMan probes and primers specific for the target and reference gene are depicted in Table 2. Relative alterations (fold change) in mRNA levels were calculated according to the Pfaffl method [50 (link)].

Cooreman A., Caufriez A., Tabernilla A., Van Campenhout R., Leroy K., Kadam P., Sanz Serrano J., dos Santos Rodrigues B., Annaert P, & Vinken M. (2022). Effects of Drugs Formerly Proposed for COVID-19 Treatment on Connexin43 Hemichannels. International Journal of Molecular Sciences, 23(9), 5018.

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Quantifying RNA Expression in Liver Cells

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Total RNA was extracted from mouse liver tissue or from human hepatoma HepaRG cells using a GenEluteTM Mammalian Total RNA purification Miniprep Kit (Sigma-Aldrich, Overijse, Belgium) and the On-column DNase I digestion Set (Sigma-Aldrich, Overijse, Belgium) according to the manufacturer’s instructions. The isolated RNA was spectrophotometrically measured using a NanoDrop^® 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) to assess purity and quantity. A cut-off ratio between 1.8 and 2.1 for the absorption at 260/280 nm was used for assessing purity. Next, the synthesis and amplification of cDNA as well as the RT-qPCR analysis were performed as explained elsewhere [76 ]. TaqMan probes and primers specific for the target and reference genes are depicted in Table 1. Relative alterations (fold change) in mRNA levels were calculated according to the 2^(−ΔΔCq) algorithm [77 (link)].

Cooreman A., Van Campenhout R., Crespo Yanguas S., Gijbels E., Leroy K., Pieters A., Tabernilla A., Van Brantegem P., Annaert P., Cogliati B, & Vinken M. (2020). Cholestasis Differentially Affects Liver Connexins. International Journal of Molecular Sciences, 21(18), 6534.

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Extracting and Quantifying RNA from Cells

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