Rnaqueous micro kit

Manufactured by Thermo Fisher Scientific

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The RNAqueous-Micro Kit is a product from Thermo Fisher Scientific designed for the isolation and purification of total RNA from small samples. The kit utilizes a specialized lysis and binding solution to capture RNA, which is then washed and eluted for downstream applications.

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RNAqueous kit (229 citations) RNAqueous-Micro (16 citations) RNAqueous-Micro isolation kit (6 citations) Ambion RNAqueous-Micro total RNA isolation kit (4 citations) RNAqueous-Micro kit RNA Isolation Procedure (3 citations) RNAqueous-Micro Total RNA kit (3 citations) Small- and micro-scale RNAqueous isolation kits (2 citations) RNAqueous-Micro" micro scale RNA isolation kit (2 citations) RNAqueous Micro kit column purification (2 citations) RNAqueous Micro-Kit and Small Scale Kit (2 citations)

437 protocols using rnaqueous micro kit

Quantifying Clock Gene Expression

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The procedures were previously described in detail¹⁸. In brief, hair follicle cells were collected before each examination by pulling out the roots of scalp hairs. Two to 10 hair follicles were quickly soaked in lysis buffer (RNAqueous‐Micro Kit; Thermo Fisher Scientific, Waltham, MA, USA). Total ribonucleic acid (RNA) was extracted and purified using an RNAqueous‐Micro Kit according to the manufacturer's instructions, and a 100 ng quantity of total RNA was reverse‐transcribed using a SuperScript VILO cDNA Synthesis Kit (Life Technologies, Carlsbad, CA, USA). The messenger RNA (mRNA) of clock genes (Dbp, E4bp4, Bmal1, Clock, Nr1d1 and Per2) was quantified by real‐time polymerase chain reaction using a Taqman MGB probe (Applied Biosystems, Waltham, MA, USA) and a 1/20 volume of the reverse transcription product. mRNA expressions were normalized using 18S rRNA expression. The relative expression value of each gene at 20.00 hours was calculated by dividing the value at 20.00 hours by the value at 0.8.00 hours for each individual.

Fujimoto R., Ohta Y., Masuda K., Taguchi A., Akiyama M., Yamamoto K., Nakabayashi H., Nagao Y., Matsumura T., Hiroshige S., Kajimura Y., Akashi M, & Tanizawa Y. (2022). Metabolic state switches between morning and evening in association with circadian clock in people without diabetes. Journal of Diabetes Investigation, 13(9), 1496-1505.

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Laser Microdissection and Transcriptomic Analysis

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For each case, the breast pathologist identified tumor areas for laser microdissection from H&E stained slides. Two to five serial sections (8 μm thick) were cut, mounted on glass PEN foil slides (Leica Microsystems, Wetzlar, Germany), stained using the LCM staining kit (Applied Biosystems, Foster City, CA) and laser microdissected on an ASLMD laser microdissection system (Leica Microsystems, Wetzlar, Germany). Slide preparation, staining and cutting were performed within a 15 min period to preserve RNA integrity. RNA was then isolated using the RNAqueous-Micro kit (Applied Biosystems, Foster City, CA) and treated with DNase I to remove any contaminating genomic DNA. RNA integrity was assessed using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), converted to biotin-labeled aRNA using two rounds of amplification with the MessageAmpII aRNA Amplification kit (Applied Biosystems, Foster City, CA), and the concentration and quality of the samples was measured with the NanoDrop 1000 (NanoDrop Products, Wilmington, DE) and 2100 Bioanalyzer. Hybridization with manufacturer provided hybridization controls, washing, staining and scanning of HG U133A 2.0 arrays (Affymetrix, Santa Clara, CA) were conducted according to manufacturer’s protocols [28 ].

Toro A.L., Costantino N.S., Shriver C.D., Ellsworth D.L, & Ellsworth R.E. (2016). Effect of obesity on molecular characteristics of invasive breast tumors: gene expression analysis in a large cohort of female patients. BMC obesity, 3, 22.

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Endothelial Cell Biology qPCR Profiling

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The RT² Profiler™ PCR Array Mouse Endothelial Cell Biology was performed using 1 μg of cDNA according to the manufacturer’s instructions (SABiosciences-Quiagen, Valencia, CA). Total RNA from pulmonary endothelial (PE) cells was purified using RNAqueous-Micro Kit (Applied Biosystems, Foster City, CA). An additional genomic DNA elimination reaction and complementary DNA (cDNA) synthesis were performed on the RNA samples using the SABiosciences RT First Strand Kit. The PCR reactions were run on a StepOnePlus Real-Time PCR Detection System (Applied Biosystems, Foster City, CA) using the following cycling parameters: one cycle of 95°C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. Product purity was verified by melting curve analysis. The ΔCt value for each gene was determined by dividing the gene Ct value by the mean Ct value of three housekeeping genes. The 2^-ΔΔCt method was used to analyze data and the change in gene expression levels were reported as fold change.

Zelko I.N., Zhu J, & Roman J. (2018). Maternal undernutrition during pregnancy alters the epigenetic landscape and the expression of endothelial function genes in male progeny.. Nutrition research (New York, N.Y.), 61, 53-63.

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Lumbosacral DRG Neuron RNA Extraction

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Lumbosacral DRG neurons (L5-S1) were homogenized using a MagNa Lyser Instrument (Roche Diagnostics, Mannheim, Germany) and 1.4 mm ceramic beads (Qiagen, Hilden, Germany)). Total RNA was extracted from L5-S1 DRG tissue using the RNAqueous^®-Micro kit (Applied Biosystems, Bedford, MA, USA). An aliquot of each RNA sample was run on a denaturing agarose gel to assess integrity. Intact RNA was then quantified by NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA), followed by treatment with DNase. DNase-treated RNA (500 ng) was reverse transcribed using the NZY M-MuLV First-Strand cDNA Synthesis Kit (NZYtech, Lisbon, Portugal), according to the manufacturer’s instructions. cDNA was diluted and stored at −20 °C for later use.

Chambel S.S., Ferreira A., Oliveira R., Miranda R., Vale L., Reguenga C., Schwab M.E, & Cruz C.D. (2022). Development of Neurogenic Detrusor Overactivity after Thoracic Spinal Cord Injury Is Accompanied by Time-Dependent Changes in Lumbosacral Expression of Axonal Growth Regulators. International Journal of Molecular Sciences, 23(15), 8667.

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RNA Isolation from Arabidopsis Shoot Samples

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All analyzed shoot samples for the 1 g controls were from seed cassettes in position 3 of each EC. For PS, position 3 corresponded to 0.76 g and for PRR position 3 corresponded to 1 g (Supplementary Figure 1). For each RNA isolation, seed cassettes were processed singly as follows. A single seed cassette was retrieved from the freezer, and the cover was removed. The cassette base was placed on a chilled platform, and RNA-later (Thermo Fisher Cat. No. AM7021) was added. The seedlings were dissected into root and shoot fractions and stored at 4°C in RNA-later for 24 hours, followed by storage at -20°C until RNA isolation. RNA was isolated from each shoot sample using the RNAqueous Micro kit (Applied Biosystems Cat No. AM1931). RNA recovery and integrity were monitored by Bioanalyzer (Agilent 2100). For the PRR shoot samples, the RNA isolation protocol was modified in order to retain the small RNA fraction as well.

Land E.S., Sheppard J., Doherty C.J, & Perera I.Y. (2024). Conserved plant transcriptional responses to microgravity from two consecutive spaceflight experiments. Frontiers in Plant Science, 14, 1308713.

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Transfection of Chicken PGCs with Anti-miR-302b

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Two weeks before the transfection, chicken FS101 and FS111 PS PGC lines were thawed. After two weeks in the culture, PGCs were collected by centrifugation and plated to 96-well plates (1000 cells/well). Next day, the cells were transfected with anti-miR-302b-5P and anti-miR-302b-3P vectors (at 100 nM final concentration) (Supplementary Table 1) (Applied Biosystems, Life Technologies, Carlsbad, US) using a siPORT™ (Applied Biosystems, Life Technologies, Carlsbad, US) transfection agent according to the manufacturer's instructions. Three 96-well plates (as biological replicates), with 6-6 parallel wells, were prepared for each condition. The proliferation rate of treated and control cells was measured using CCK-8 reagent (1 : 10, Dojindo Laboratories, Japan) every day, for 3 days. For detailed RNA expression analysis, cells were harvested in lysis buffer of RNAqueous®-Micro Kit (Applied Biosystems, Life Technologies, Carlsbad, US) 48 hours following the transfection.

Lázár B., Anand M., Tóth R., Várkonyi E.P., Liptói K, & Gócza E. (2018). Comparison of the MicroRNA Expression Profiles of Male and Female Avian Primordial Germ Cell Lines. Stem Cells International, 2018, 1780679.

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Quantitative Gene Expression Analysis

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Total RNA was prepared from purified PEC and PFTC cells using RNAqueous-Micro Kit (Applied Biosystems, Foster City, CA). The synthesis of single stranded DNA from RNA was performed using SuperScript First-Strand Synthesis System for RT-PCR and random hexamers (Invitrogen, Carlsbad, CA), according to the protocol provided by manufacturer. To quantitate the abundance of gene-specific mRNAs, quantitative PCR was undertaken using the StepOnePlus Real-Time PCR Detection System (Applied Biosystems, Foster City, CA) and an SYBR® Green Master Mix. The PCR cycles were 95 °C for 3 min, then 40 cycles of 95 °C for 15 s, 60 °C for 1 min. The nucleotide sequences of primers used for qRT-PCR are presented in Table 1. PCR assays were run in triplicate, and gene expression was normalized to P-Actin mRNA levels.

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Osteogenic Gene Expression Analysis

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The expression of ALP, Col1A1, DMP-1, and DSPP was assessed by real-time PCR after 14 d of culture (n=4). For RNA extraction and cDNA synthesis, the RNaqueous® Micro Kit (Applied Biosystems, Foster City, CA, USA) and High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), respectively, were used following the manufacturer's recommendations. Relative gene expression was analyzed on a StepOnePlus™ Real-Time PCR System (Applied Biosystems) using the TaqMan assay (Applied Biosystems) according to the manufacturer's protocol. The β-actin gene was used as a constitutive gene. Data were calculated according to the 2ΔΔCT equation using the CHAlCa group for data normalization.

Cassiano F.B., Soares D.G., Bordini E.A.F., Anovazzi G., Hebling J, & Costa C.A.S. (2020). Simvastatin-Enriched Macro-Porous Chitosan-Calcium-Aluminate Scaffold for Mineralized Tissue Regeneration. Brazilian dental journal, 31(4).

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Quantification of Nasal IL-33 mRNA

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IL-33 mRNA was determined using the nasal mucosa samples of patients as described previously (11) . Patient information, preparations for scraping the nasal mucosa, evaluation of nasal symptoms, and other experimental conditions were previously reported (3). Blood samples were collected and blood cell analysis was conducted to count eosinophils. Nasal mucosa scrapings were obtained from patients by an otolaryngologist as previously described (3) . Total RNA was isolated and reverse transcribed using the RNAqueous Micro Kit (Applied Biosystems) and the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), respectively. The nucleotide sequences of the primers and probes for human IL-33 are listed in Table 2. Universal Probe Library #27 (Roche Diagnostics, Basel, Switzerland) were used for the IL-33 probes. The pumilio RNA binding family member 1 (PUM1) primer and probe kit (Hs 00206469 -m1, Applied Biosystems) was used to generate a standard (3) . The data are expressed as the ratio of IL-33 mRNA to PUM1 mRNA as previously described. The ethics committee of Tokushima University Hospital and Yashima General Hospital approved this study ; written informed consent was obtained from each patient before the study commenced.

Islam R., Mizuguchi H., Shaha A., Nishida K., Yabumoto M., Ikeda H., Fujino H., Kitamura Y., Fukui H, & Takeda N. (2018). Effect of wild grape on the signaling of histamine H₁ receptor gene expression responsible for the pathogenesis of allergic rhinitis. The journal of medical investigation : JMI, 65(3.4).

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Robust RNA-Seq Library Preparation

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RNA isolation was performed following RNAqueous Micro kit (Life Technologies) directions, including optional DNAse treatment. SoLo RNA-Seq System (NuGEN) was used to construct cDNA libraries, following manufacturer’s directions. Libraries were screened for quality on a Bioanalyzer (Agilent). No more than 50% of the NuGEN libraries were pooled with other TruSeq libraries to maintain the sequence diversity. Using TruSeq SR Cluster Kit v3, at 15 pM the libraries were clustered onto the flow cell and sequenced on HiSeq 1000 (Illumina) using Illumina standard sequencing primer. At single read 1× 51 bp setting, at least 45 million reads per sample were generated.

Serafin E.K., Chamessian A., Li J., Zhang X., McGann A., Brewer C.L., Berta T, & Baccei M. (2019). Transcriptional profile of spinal dynorphin-lineage interneurons in the developing mouse. Pain, 160(10), 2380-2397.

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