Total RNA was isolated from melanoma tissues using the PerfectPure RNA Tissue kit (5Prime Inc., MD, USA) at the University of Pittsburgh Cancer Institute (UPCI). RNA was quantified using Ribogreen RNA quantitation Kit (Molecular Probes, Eugene OR). RNA quality was also evaluated by RNA integrity number using the Agilent Bioanalyzer.
Ribogreen rna quantitation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RiboGreen RNA Quantitation Kit is a fluorescence-based assay that allows for the sensitive and accurate quantitation of RNA in solution. The kit uses a proprietary fluorescent dye that binds to RNA, enabling the measurement of RNA concentrations across a wide linear range. The kit provides a simple and reliable method for determining RNA levels in biological samples.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Tissuelyser 2, by Qiagen (2 mentions)
Agilent bioanalyzer, by Agilent Technologies (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Rneasy column, by Qiagen (1 mentions)
Pidt blue vector, by Integrated DNA Technologies (1 mentions)
Mmessage in vitro transcription kit, by Thermo Fisher Scientific (1 mentions)
Pgem t easy, by Promega (1 mentions)
Infinium humanmethylation27 beadchip, by Illumina (1 mentions)
Ht 12 expression beadchip, by Illumina (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Spectrafluor microplate reader, by Tecan (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Mmessage mmachine t7 kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Genechip mouse genome 430 2.0 array, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
9 protocols using ribogreen rna quantitation kit
1
Melanoma Tissue RNA Isolation
2
RNA Fragment Preparation for Viral Detection
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Synthetic RNA fragments of the viruses were prepared for LASV, RVFV, and YFV, MARV and CCHFV [24 (link)]. The target regions of LASV, RVFV and YFV viruses to be detected were amplified by RT-PCR, purified using QIAquick PCR purification kit (Qiagen, Inc., Valencia, CA), and cloned into the expression vector pGEM T Easy (Promega, Madison, WI) containing the T7 promoter region. The plasmids were purified, and the presence of complete inserts was confirmed by sequencing the inserts using vector-specific primers. Target regions for MAR and CCHF were synthetically constructed and inserted into the pIDT Blue vector (Integrated DNA Technologies Coralville, IA). The complete inserts of the target regions thus generated were linearized and in vitro transcribed using the mMessage in vitro transcription kit (Ambion, Austin, TX). Following DNAse treatment, the synthetic RNA was purified using an RNeasy column (Qiagen, Inc., Valencia, CA). The quantity of RNA generated for each transcript was determined using the Ribogreen® RNA Quantitation Kit (Molecular Probes, Eugene, OR) following the manufacturer’s instructions.
3
Comprehensive Molecular Profiling of Melanoma
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Total RNA was isolated from melanoma tissues using the PerfectPure RNA Tissue kit (5Prime Inc., MD, USA). RNA was quantified using Ribogreen RNA quantitation Kit (Molecular Probes, Eugene OR) and its quality was evaluated by RNA Integrity Number using the Agilent Bioanalyzer. Whole genome gene expression analysis was carried out using the Illumina HT-12 Expression BeadChip (Illumina, San Diego, CA) which targets > 25,000 genes (>48,000 probes) derived from the RefSeq (Build 36.2, Rel 22) and UniGene databases7 (link).
DNA was isolated from melanoma tissues using ArchivePure DNA Cell/Tissue kit (5Prime Inc., MD, USA). DNA samples (0.5 μg) were treated with sodium bisulphite using the EZ DNA methylation Gold kit (Zymo Research, Irvine, CA), and bisulphite-treated DNA was applied to an Illumina Infinium HumanMethylation27 BeadChip (Illumina, San Diego, CA) for DNA methylation profiling. This microarray permits the quantitative measurement of DNA methylation for 27,578 CpG dinucleotides spanning 14,495 genes. Methylation status of the interrogated CpG sites was determined by comparing β-values, the ratio of the fluorescent signal from the methylated allele to the sum from the fluorescent signals of both methylated and unmethylated alleles.
DNA was isolated from melanoma tissues using ArchivePure DNA Cell/Tissue kit (5Prime Inc., MD, USA). DNA samples (0.5 μg) were treated with sodium bisulphite using the EZ DNA methylation Gold kit (Zymo Research, Irvine, CA), and bisulphite-treated DNA was applied to an Illumina Infinium HumanMethylation27 BeadChip (Illumina, San Diego, CA) for DNA methylation profiling. This microarray permits the quantitative measurement of DNA methylation for 27,578 CpG dinucleotides spanning 14,495 genes. Methylation status of the interrogated CpG sites was determined by comparing β-values, the ratio of the fluorescent signal from the methylated allele to the sum from the fluorescent signals of both methylated and unmethylated alleles.
4
Quantification of Nucleic Acids by Fluorescence
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Nucleic acids (RNA and DNA) were quantified following the procedure described in Varó et al.
(2007), using RiboGreen™ RNA Quantitation Kit and PicoGreen™ DNA Quantitation Kit, respectively (Molecular Probes). Briefly, samples were homogenised in 1 mL ice-cold TE buffer (10 mM Tris-HCl buffer containing 1 mM EDTA, pH = 7.5). After centrifugation (10,000 g, 10 min, 4°C) the supernatant was transferred to a clean tube for the analyses of nucleic acids and proteins. For RNA and DNA determinations 50 μL of diluted supernatant were transferred in triplicate into a 96-well black microplate containing 1 μL of DNase I or 10 μL of RNase A (diluted 1:400), respectively. After 1 h incubation at 37°C, the volume was adjusted to 100 μL with TE buffer and 100 μL of RiboGreen or PicoGreen was added for RNA and DNA quantification, respectively, and allowed to stain for 5 min in darkness before reading in a TECAN SPECTRA-FLUOR microplate reader, at 485 nm EX/535 nm EM (TECAN, Salzburg, Austria). Concentrations were calculated from high-range standard curves of RNA (20ng•mL -1 -1μg•mL -1 ) or DNA (1ng•mL -1 -1μg•mL -1 ) prepared from standards supplied with Ribo-Green and Pico-Green reagent kits.
(2007), using RiboGreen™ RNA Quantitation Kit and PicoGreen™ DNA Quantitation Kit, respectively (Molecular Probes). Briefly, samples were homogenised in 1 mL ice-cold TE buffer (10 mM Tris-HCl buffer containing 1 mM EDTA, pH = 7.5). After centrifugation (10,000 g, 10 min, 4°C) the supernatant was transferred to a clean tube for the analyses of nucleic acids and proteins. For RNA and DNA determinations 50 μL of diluted supernatant were transferred in triplicate into a 96-well black microplate containing 1 μL of DNase I or 10 μL of RNase A (diluted 1:400), respectively. After 1 h incubation at 37°C, the volume was adjusted to 100 μL with TE buffer and 100 μL of RiboGreen or PicoGreen was added for RNA and DNA quantification, respectively, and allowed to stain for 5 min in darkness before reading in a TECAN SPECTRA-FLUOR microplate reader, at 485 nm EX/535 nm EM (TECAN, Salzburg, Austria). Concentrations were calculated from high-range standard curves of RNA (20ng•mL -1 -1μg•mL -1 ) or DNA (1ng•mL -1 -1μg•mL -1 ) prepared from standards supplied with Ribo-Green and Pico-Green reagent kits.
5
Expressing Slo2.1, Kir6.2, and SUR1 in Oocytes
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cDNA for human Slo2.1 (KCNT2, NCBI Genbank accession no. NM_198503), kindly provided by L. Kaczmarek (Yale University), was subcloned into the psGEM oocyte expression vector as described (Dai et al. 2010 (link)). K1031A Slo2.1 was generated by using the QuikChange site‐directed mutagenesis kit (Agilent Technologies) and confirmed by DNA sequencing by the University of Utah sequencing core facility. Human Kir6.2 (KCNJ11) and rat SUR1 (ABCC8) cDNAs in the pBF vector were kindly provided by F. Ashcroft (University of Oxford). Plasmids were linearized with SfiI (Slo2.1) or Mlu1 (Kir6.2, SUR1). cRNAs were prepared using the mMessage mMachine T7 kit (Slo2.1) or SP6 kit (Kir6.2, SUR1) (Ambion, Life Technologies, Grand Island, NY). Concentrations of cRNA were measured with the RiboGreen RNA quantitation kit (Invitrogen, Life Technologies).
6
Adipose Tissue Gene Expression Profiling
7
Profiling mTEC and mNEC Transcriptomes
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Total RNA was extracted from three types of mTEC (melanoma-derived EC, renal carcinoma-derived EC and oral carcinoma-derived EC) and mNEC using TRIzol (Invitrogen) according to the manufacturer's standard protocol. RNA was quantified using a RiboGreen RNA Quantitation Kit (Invitrogen) and RNA quality was confirmed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Gene expression profiles were obtained from 1.5 μg total RNA per sample using a GeneChip Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA, USA) according to the manufacturer's recommended protocol (GeneChip 3'-IVT Express Kit, P/N 702646 Rev. 7).
8
Total RNA Extraction and DWV Quantification
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Total RNA extraction and DWV absolute quantification were performed as previously described [18 (link)]. Briefly, individual heads and abdomens were separately homogenized using a TissueLyser II (Qiagen), total RNA was extracted with RNeasy mini Kit (Qiagen), eluted in 30 μl RNase-free water and quantified with RiboGreen RNA Quantitation Kit (Invitrogen, Carlsbad, CA, USA). Ten microliters of each RNA were used as template to determine the viral load by RT-qPCR. Samples with RT-qPCR amplification signal were considered positive and those without signal negative. Results were expressed as viral copy number per microgram of RNA.
9
Bee and Pollen RNA Extraction
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Bee samples were homogenized using a TissueLyser II (Qiagen, Hilden, Germany) for 3 min. at 25 Mhz, total RNA was extracted with RNeasy Kit (Qiagen), eluted in 30 µl RNase-free water, quantified with RiboGreen RNA Quantitation Kit (Invitrogen, Carlsbad, CA, USA) and stored in aliquots at −80°C. Pollen samples (20 mg each) were suspended in 140 µl PBS, vortexed for 30 s and centrifuged at 14000 rpm for 5 min. Supernatant was used for RNA extraction with QIAamp Viral RNeasy Kit (Qiagen). RNA was eluted in 30 µl RNase-free water, aliquoted and stored at −80°C.
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