Yeast ribosomes were prepared as previously described26 ,27 (link). Briefly, cell pellets were washed with ice-cold water and resuspended in buffer (10 mM MgCl2, 100 mM KCl, 50 mM Tris/HCl, pH 7.5, 0.4 mM PMSF) at 4 °C. Equal volumes of glass beads (400 μm in diameter) were added, and cells were broken using 8 pulses of vortexing (30 sec each) punctuated by cooling on ice. Cell debris was precipitated at 11,300 × g for 2 min at 4 °C in F-34-6-38 Eppendorf rotor. Lysate was further clarified by centrifugation at 11.300 × g for 10 min at 4 °C in F-34-6-38 Eppendorf rotor. After clarification, 1/10 of the total lysate volume was used to isolate total cellular RNA (S30). Subsequently, ribosomes were pelleted (P100) from lysates by centrifugation at 160,000 × g for 90 min at 4 °C in Beckman 70.1 Ti rotor and suspended in the storage buffer (2 mM Mg(OAc)2, 100 mM KOAc, 20 mM HEPES, pH 7.4, 0.1 mM PMSF, 1 mM DTT, 20% glycerol). The top two-thirds of the post-ribosomal supernatant were collected and frozen, and designated as the S100 fraction. P100, S100 and S30 fractions were mixed with TRI Reagent (MRC), flash frozen in liquid nitrogen and subjected to RNA isolation following the manufacturer’s instructions. The purity of P100 and S100 fraction was verified with Agilent Bioanalyzer 2100 with the use of RNA Nano 6000 kit.
Rna 6000 nano
Manufactured by Agilent Technologies
Sourced in United States, France
The RNA 6000 Nano is a lab equipment product by Agilent Technologies designed for the analysis of RNA samples. It provides quantification and sizing of RNA molecules in the range of 25-6000 nucleotides.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (23 mentions)
Trizol reagent, by Thermo Fisher Scientific (14 mentions)
2100 bioanalyzer, by Agilent Technologies (10 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (8 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Tripure isolation reagent, by Roche (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Mirneasy mini kit, by Qiagen (4 mentions)
Rneasy kit, by Qiagen (3 mentions)
Novaseq 6000, by Illumina (3 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Small rna nano labchip, by Agilent Technologies (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Nanophotometer, by Implen (2 mentions)
Rnalater, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Rna 6000 pico kit, by Agilent Technologies (2 mentions)
61 protocols using rna 6000 nano
1
Isolation and Purification of Yeast Ribosomes
2
RNA-seq Analysis of Breast Cancer
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After assessing RNA integrity using RNA Nano 6000 (Agilent Technologies, CA, USA). RNA samples of clinical BC tissues or FOXD1 knockdown and control cell lines were subjected to library construction and then sequenced on Illumina Novaseq 6000. FeatureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. RNA sequencing technology was provided by Novogene (Beijing, China).
3
RNA Isolation and Purification Protocol
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RNA was isolated from cells using the Trizol reagent (ThermoFisher) and purified according to the manufacturer’s protocol. All purified RNA was subsequently treated with DNaseI digestion to remove possible DNA contaminants (Qiagen). The quality of RNA used for cDNA library preparation was verified using the RNA nano 6000 analysis chip on a BioAnalyzer 2000 series instrument (Agilent Technologies) to ensure an RNA integrity value greater than or equal to 9.
4
RNA and Protein Extraction Protocol
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Total RNA and proteins were extracted using TriPure Isolation Reagent (Roche). RNA integrity (RIN) was determined using RNA Nano 6000 (Agilent). The RIN was 8.5 ± 0.5. RNA was quantified using a NanoDrop 2000 spectrophotometer (ThermoFischer, Spain).
5
RNA-seq Analysis of NMDA-Induced Retinal Degeneration
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The mouse retinas were collected 5 days after NMDA intervention. Three individual retinas were treated as one sample, and each group contained 3 samples. RNA was isolated by total RNA kit (R6834-01, Omega Bio-Tek),. Total amounts and integrity of RNA were assessed using the RNA Nano 6000 assay kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). After RNA was converted to cDNA, the samples were sequenced by the Illumina NovaSeq 6000 at Novogene (Beijing, China). Genes with a fold-change ≥1.5 identified by edgeR and a false discovery rate <0.05 were considered differentially expressed (BMKCloud, http://www.biocloud.net/ ). Gene functional annotations were based on the Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.genome.jp/kegg/ ) and Gene Ontology (GO, http://www.geneontology.org/ ) databases.
6
Transcriptional Profiling of Caecum in Nlrp12 Knockout
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Caecum specimens from non-infected and infected wild-type and Nlrp12−/− animals were dissected out and stored at −80 °C in RNAlater® (Ambion, Applied Biosystems, Foster City, CA), until extraction of total RNA accordingly to manufacturer’s instructions (Qiagen). The quality of the extracted RNA was confirmed by Agilent 2100 Bioanalyzer using RNA Nano 6000 (Agilent Technologies). The 4x44K Whole Mouse Genome Oligo Microarrays (Agilent Technologies) was used to determine the gene expression profile of two biological replicates. For each labeling, 2 µg of total RNA per sample were engaged in the synthesis of a fluorescent probe labeled with Cy5 or Cy3 fluorophores. A 2 × 2 factorial experimental design and a dye-swap strategy were used (GEO accession number GSE59940). After a within array loess normalization, raw data were analyzed with the LIMMA package and sets of differentially expressed genes were filtered for a p-value <0.01 and a limit log fold change >1 by using moderated t-statistic with empirical Bayes shrinkage of the standard errors. Statistics were corrected for multiple testing using False Discovery Rate approach. A gene-ontology analysis using Panther was next performed on up- and downregulated genes that are referred in Unigene (http://www.pantherdb.org/ ).
7
Tissue RNA and Protein Extraction
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Total RNA and proteins were sequentially extracted from cells and human or mouse hippocampal tissue using Tripure Isolation Reagent (Roche, Basel, Switzerland; 100 mg tissue/1 ml Tripure Isolation Reagent) following the manufacturer's recommendations. RNA integrity (RIN) was determined by RNA Nano 6000 (Agilent, Santa Clara, CA). Although no differences between Braak groups were observed, the RIN was lower in human samples compared with transgenic models (RIN: 4.95 ± 1.4 or 8.5 ± 0.5 for human and mouse samples, respectively). RNA was quantified using NanoDrop 2000 spectrophotometer (Thermo Fisher, Waltham, MA). Proteins were quantified using Lowry's method.
8
RNA Isolation and Quantification for qPCR
9
RNA and Protein Extraction Protocol
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Total RNA and proteins were extracted using TriPure Isolation Reagent (Roche) [22 (link)]. RNA integrity (RIN) was determined by RNA Nano 6000 (Agilent). Although no differences between Braak groups were observed, the RIN was lower in human samples compared with transgenic models (RIN: 4.95 ± 1.4 or 8.5 ± 0.5 for human and mouse samples, respectively). RNA was quantified using NanoDrop 2000 spectrophotometer (Thermo Fischer). Proteins were quantified using Lowry’s method.
10
RNA Extraction and RNA-seq Pipeline
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RNA was extracted following the method of Clark, Acorn, et al. (2013 ) and RNA‐seq performed on a total of 11 animals drawn from the four treatment combinations (n = 3 in all treatments except the control 16°C, 400 ppm treatment, where n = 2). Briefly, the preserved postlarvae were individually homogenized in 1 ml Trizol (TH electric homogenizer; OMNI International), placed in a chloroform/Trizol mixture (200 µl chloroform/ml of Trizol), and incubated for 3 min at room temperature. Samples were then centrifuged at 12,000 g at 4°C (15 min), and the collected supernatant was added to an equal volume of 100% ethanol. RNA was extracted with a RNeasy kit (Qiagen) with an on‐column DNasel digestion, and quantified using a NanoDrop1000 spectrophotometer (Thermo Fisher Scientific). Quality was verified with the Agilent Bioanalyzer 2100 and RNA Nano 6000 chips.
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