Total RNA was isolated using TRIzolTM Reagent (Invitrogen, United States) according to manufacturer’s instructions and treated with DNAse I, RNAse-free (Thermo Fisher, United States). The RNA concentration was quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher). RNA integrity was determined via agarose electrophoresis and by employing the Agilent Bioanalyzer platform 2100 (Agilent, United States).
Agilent 2100 bioanalyzer platform
Manufactured by Agilent Technologies
Sourced in United States, Germany
The Agilent 2100 Bioanalyzer platform is a microfluidics-based lab-on-a-chip system designed for the analysis of nucleic acids and proteins. It provides automated, quantitative, and qualitative analysis of samples in a compact, easy-to-use format.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (12 mentions)
Trizol reagent, by Thermo Fisher Scientific (11 mentions)
Rneasy mini kit, by Qiagen (6 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Agilent rna 6000 nano kit, by Agilent Technologies (4 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Novaseq 6000, by Illumina (3 mentions)
Agilent gene expression hybridization kit, by Agilent Technologies (3 mentions)
Hiseq 2000, by Illumina (2 mentions)
Dna free kit, by Thermo Fisher Scientific (2 mentions)
Qiazol lysis reagent, by Qiagen (2 mentions)
Rneasy lipid tissue mini kit, by Qiagen (2 mentions)
Tissueruptor, by Qiagen (2 mentions)
Mirneasy kit, by Qiagen (2 mentions)
Agilent whole human genome microarrays 4 44 k, by Agilent Technologies (2 mentions)
Ribo zero magnetic kit, by Illumina (2 mentions)
Qiaquick pcr isolation kit, by Qiagen (2 mentions)
Truseq stranded mrna sample preparation kit, by Illumina (2 mentions)
Agilent s microarray scanner system, by Agilent Technologies (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
50 protocols using agilent 2100 bioanalyzer platform
1
Total RNA Isolation and Characterization
2
RNA Sequencing of Fungal Strains
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Total RNA was isolated using TRIzol Reagent (Invitrogen, USA) according to the manufacturer's instructions and treated with DNase I, RNase-free (Thermo Fisher, USA). The RNA concentration was quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher). The RNA integrity was determined by agarose formaldehyde gel electrophoresis and using the Agilent Bioanalyzer platform 2100 (Agilent, USA). Purity and concentration were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher). A total of 12 libraries (Δmus-52 and Δpac-3 strains, cultivated in high- and low-Pi concentration media, each, in three biological replicates) were sequenced on an Illumina HiSeq2000 (Illumina, USA) sequencer platform with paired-end 100-bp reads. RNA-seq data were analyzed and validated as previously described (Martins et al., 2019 (link)) and deposited at the GEO database with accession number GSE132373.
3
Transcriptome Profiling by RNA-Seq
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RNA-Seq libraries were prepared using the Kapa RNA HyperPrep Kit with RiboErase (Kapa Biosystems, Cat# KR1351) according to the manufacturer's protocol. Twenty five ng of total RNA from each sample was subjected to ribosomal RNA depletion. The hybridization mix was purified and treated with DNase to remove the hybridization oligonucleotides. Purified RNA underwent fragmentation, first-strand cDNA synthesis, and second-strand cDNA synthesis, followed by 3′ end adenylation. Barcoded adaptors were ligated to the adenylated, double-stranded cDNA fragments. 13 cycles of PCR were performed to produce the final sequencing library. cDNA library quality was determined using a High Sensitivity DNA Chip (Agilent, Cat# 5067–4626) with the Agilent 2100 Bioanalyzer platform, and cDNA libraries were quantified using a Qubit fluorometer (Thermo Fisher Scientific). Library templates were prepared for sequencing using the HiSeq SR Cluster v4 Kit (Illumina, Cat# GD-401-4001). Sequencing runs were performed using the Illumina HiSeq 2500 platform with HiSeq SBS v4 Kit (Illumina, Cat# FC-401-4002). The HiSeq Control (HCS 2.2.38) and Real-Time Analysis (RTA 1.18.61) software were used for image analysis and base calling.
4
Total RNA Extraction from MCF7 Cell Lines
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Before and after docetaxel treatment for 24 h, total RNA was extracted from the MCF7pcNEO and MCF7pcPAR4 cells using the acid guanidinium thiocyanate-phenol-chloroform method (30 (link)). The RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The sample integrity was also determined by RNA Nano Chips using the Agilent 2100 Bioanalyzer platform (Agilent Technologies, Inc., Santa Clara, CA, USA). High quality RNA was purified using the RNeasy Mini kit (Qiagen, Germantown, MD, USA).
5
Hippocampal RNA Isolation and Analysis
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RNA isolation was performed as previously described [51] (link). In brief, hippocampi were put into 1 ml QIAzol Lysis reagent (Qiagen) and immediately homogenized by a rotor-stator homogenizer (TissueRuptor, Qiagen). RNA was isolated using the RNeasy Lipid Tissue Mini Kit (Qiagen), following the manufacturers protocol. To remove contaminating DNA, 20 µl isolated RNA was treated with DNase using the DNA-free Kit (Ambion, Carlsbad, CA, USA). RNA quality and quantity were determined on the Agilent 2100 Bioanalyzer platform (RNA 6000 Nano, Agilent Technologies, Waldbronn, Germany) and validated on the NanoDrop device (NanoDrop, Wilmington, USA). Only RNA with sufficient quality (RIN>8) and purity (OD260/OD280 nm absorption ratio >1.95) were further used. Absence of genomic DNA and unspecific binding of primers was confirmed in qPCR by not reaching the threshold after 40 cycles in samples without reverse transcription.
6
Liver RNA Isolation Protocol
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Livers were removed from sacrificed mice, rapidly frozen in liquid nitrogen and stored at −80°C until use. Frozen livers were grounded in a mortar under liquid nirogen and aliquots were used to isolate total RNA using Trizol (Sigma-Aldrich). An additional cleaning step was performed by using the miRNeasy Kit (Qiagen). RNA quality and integrity were determined using the Agilent RNA 6000 Nano Kit on the Agilent 2100 Bioanalyzer platform (Agilent Technologies). RNA was quantified by measuring A260 nm on the ND-1000 Spectrophotometer (NanoDrop Technologies).
7
Comprehensive RNA Extraction from Mouse Livers
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Livers were aseptically removed from sacrificed mice, rapidly frozen in liquid nitrogen and stored at −80°C until use. Frozen livers were grounded in a mortar under liquid nirogen and aliquots of each liver were used to isolate total RNA by the Trizol (Qiagen, Hilden, Germany) standard RNA extraction protocol. Trizol-extracted RNA was additionally cleaned up using the miRNeasy Kit (Qiagen). Integrity and quality of RNA was checked with the Agilent 2100 Bioanalyzer platform (Agilent Technologies). All RNA samples revealed RIN values between 8.7 and 9.1.
8
Transcriptome Profiling of Plant Root Tissues
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For transcriptome profiling, high-quality total RNA was isolated from frozen root tissues from two biological replicates using the RNeasy® Plant Mini Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Genomic DNA contamination was eliminated by incubating the samples with RNase-free DNase (1 U μl-1) at 37°C for 15 min, and then at 65°C for 10 min. RNA quality was tested by measuring the A260/A280 and A260/A230 ratios using a NanoDrop Spectrophotometer. Electropherograms were obtained using an Agilent 2100 Bioanalyzer platform (Agilent Technologies, USA) with an Agilent RNA 6000 Nano Kit; Agilent 2100 Expert software version B.02.03.SI307 was used to calculate the RNA integrity number (RIN) [53 (link)]. RIN values of samples ranged from 7.0 to 7.5. A 1 μg aliquot of total RNA was subjected to mRNA enrichment using the Dynabeads® mRNA DIRECT™ Micro Kit (Cat. nr. 61021, Life Technologies) according to the manufacturer’s instructions.
9
RNA Extraction from Blood, Serum, and CSF
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Peripheral blood samples have been obtained from MCI-AD and AD patients. For RNA isolation and purification from whole blood, the Trizol (Invitrogen, #15596-026) method has been used for all samples within 1 h from drawing, thus reducing RNA degradation. The RNA isolated with this protocol comes from all white cells, including polymorphonuclear leukocytes and mononuclear cells. RNA was isolated including an optional DNase digestion step. This standardized RNA isolation procedure guarantees high-quality non-degraded RNA. RNA samples were quality-checked by the identification of 18S rRNA and 28S rRNA peaks via the Agilent 2100 Bioanalyzer platform (Agilent Technologies). Serum and CSF samples were also extracted from all MCI-AD and AD patients, centrifuged at 12,000 × g for 5 min at 4 °C and stored at − 80 °C for total RNA extraction using Trizol reagent (Invitrogen). Finally, with the same procedures outlined above, serum samples from healthy controls were extracted and processed.
10
RNA Isolation and Microarray Analysis
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Samples were shipped to Miltenyi Biotec GmbH for RNA isolation and microarray analysis. RNA was isolated using Trizol, and quality control on total isolated RNA was performed using the Agilent 2100 Bioanalyzer expert software that allows for visual control and generation of an RNA integrity Number (RIN) for integrity and overall quality of the samples (36 (link)). SuperAmp RNA amplification was performed according to Miltenyi Biotec’s procedure. Briefly, the amplification is based on a global PCR protocol using mRNA-derived cDNA. mRNA was isolated via magnetic bead technology. Amplified cDNA samples were quantified using the ND-1000 Spectrophotometer (NanoDrop) showing a consistent 260/280 ratio of 1.8 across all samples. The integrity of the cDNA was checked via the Agilent 2100 Bioanalyzer platform (Agilent Technologies). The results of the Bioanalyzer run are visualized in a gel image and an electropherogram using the Agilent 2100 Bioanalyzer expert software. The average length of the highly amplified cDNA products ranged between 200–1,000 bp.
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