For in vitro RNA extraction, exponential phase C. glabrata cells were centrifuged and the pellet was snap-frozen in liquid nitrogen and further processed similarly to organs. RNA from organs was extracted similarly according to Amorim-Vaz et al. [30 (link)]. Briefly, organs were removed from RNAlater, snap-frozen in liquid nitrogen and subsequently mashed using a mortar. The resulting powder was resuspended in RNA buffer (0.1 M TrisHCl pH 7.5, 0.1 M LiCl, 10 mM EDTA, 0.5% SDS), TrizolReagent (Ambion) and phenol-chlorophorm-isoamylalcohol (PCI) (Sigma). Acid-washed glass beads (Sigma) were added and mechanical lysis was carried out in the Precellys Evolution instrument (Bertin Technologies). Upon centrifugation, the supernatant was mixed with PCI, vortexed and centrifuged. The resulting supernatant was then mixed with 100% ethanol and further processed with the Directzol RNA MiniPrep kit (Zymo Research) according to instructions. RNA quality was verified using a 5200 Fragment Analyser (Agilent) upon preparation with the high sensitivity RNA kit (Agilent).
5200 fragment analyser
Manufactured by Agilent Technologies
Sourced in United States
The 5200 Fragment Analyzer is a lab equipment product offered by Agilent Technologies. It is a high-performance capillary electrophoresis system designed for the analysis of DNA, RNA, and protein fragments. The 5200 Fragment Analyzer provides accurate sizing and quantification of nucleic acid and protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Direct zol rna miniprep kit, by Zymo Research (1 mentions)
Rna high sensitivity kit, by Agilent Technologies (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Acid washed glass beads, by Merck Group (1 mentions)
Precellys evolution, by Bertin Technologies (1 mentions)
Nanodrop one, by Thermo Fisher Scientific (1 mentions)
Accuprep universal rna extraction kit, by Bioneer (1 mentions)
Accupower rocketscript cycle rt premix, by Bioneer (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Sera mag oligo dt magnetic beads, by GE Healthcare (1 mentions)
Kapa dual indexed adapter kit, by Roche (1 mentions)
Surveyor mutation detection kit, by Integrated DNA Technologies (1 mentions)
Direct zol microprep kit, by Zymo Research (1 mentions)
E220 focused ultrasonicator, by Covaris (1 mentions)
Dnf 471 standard sensitivity rna reagent kit, by Agilent Technologies (1 mentions)
Ovation rna seq system for drosophila, by Tecan (1 mentions)
5 protocols using 5200 fragment analyser
1
RNA Extraction from C. glabrata and Organs
2
Liver mRNA Expression Analysis in Mice
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Eight‐week‐old C57BL/6 mice were administered Fa EVs (50 μg) by intraperitoneal injection. One hour postinjection, liver mRNA was extracted using an AccuPrep Universal RNA Extraction Kit (Bioneer, Daejeon, South Korea), and the purity and quality of mRNA were measured by a NanoDrop One (Thermo Fisher Scientific Inc.) and 5200 Fragment Analyser (Agilent Technologies, Inc. Santa Clara, CA, USA) according to the Minimum Information for Publication of Quantitative Real‐Time PCR Experiments (MIQE) guidelines. Complementary DNA was synthesized using AccuPower Rocketscript Cycle RT Premix (Bioneer). qPCR array was performed as previously described (Ahn et al., 2016 (link); Hong et al., 2021 (link)). Data analysis was based on the 2−ΔΔCt method. Average threshold cycles (Ct) for the gene of interest were obtained from triplicate samples and normalized by the Ct of HPRT as a housekeeping gene. The gene information is listed in Table S2 .
3
Illumina-based mRNA Sequencing Library Prep
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Illumina-based mRNA sequencing libraries were prepared from 1 μg RNA following Finkel and colleagues [61 (link)]. Briefly, mRNAs were selected using Sera-mag oligo(dT) magnetic beads (GE Healthcare Life Sciences). RNAs were washed and fragmented at 94°C for 6 minutes. First-strand cDNA synthesis was performed using random hexamers and reverse transcriptase (Superscript III reverse transcriptase, Invitrogen, Carlsbad, CA). Second-strand cDNA synthesis was done using DNA Polymerase I and RNAseH. Double-stranded cDNAs were end-repaired using T4 DNA polymerase, T4 polynucleotide kinase, and Klenow polymerase. The DNA fragments were then adenylated using Klenow exo-polymerase to allow the ligation of Illumina adapters (Kapa Dual-indexed adapter kit, Roche, Basel, Switzerland). Unless specified, reagents were purchased from Enzymatics. Library quality control and quantification were performed using the 5200 Fragment Analyser and the NGS fragment kit (Agilent Technologies, Santa Clara, CA). Libraries were sequenced using Illumina HiSeq4000 sequencers to generate 50-bp single-end reads.
4
Detecting Genetic Mutations via Surveyor Assay
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The presence of a mutation in the amplified target sequence was detected using the Surveyor Mutation Detection Kit (#706021, Integrated DNA Technologies). Two PCR products (a reference product and a sample to be analysed) were mixed in a 10 μL final volume and the endonuclease mismatch specific cleavage was performed following the manufacturer recommendations. The mixture was then loaded on capillary electrophoresis system (Fragment Analyser 5200, Agilent or GXII, PerkinElmer) prior to analysis of the cleavage profile. When needed for confirmation of the presence of mutations, the PCR products were sequenced using the Sanger method with the forward and reverse primers used for the RT-PCR (Genewiz).
5
Drosophila Brain Transcriptome Analysis
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For each sample, five male third instar larvae brains were homogenised and RNA was purified using the Directzol microprep kit (Zymo research) and using the standard protocol with on-column DNase treatment. RNA quality was determined with a Fragment Analyser 5200 (Agilent Technologies, Inc.) using the DNF-471 Standard Sensitivity RNA reagent kit.
Total-RNA libraries were synthesised using the Ovation RNA-Seq system for Drosophila (NuGEN) . Standard protocol procedure with integrated DNase treatment was used to make the sequencing libraries. Fragmentation was performed using a Covaris E220 Focusedultrasonicator with the recommended settings for 200 base pair target length. The library quality was evaluated on a Fragment Analyser using the DNF-920 DNA reagent kit.
Total-RNA libraries were synthesised using the Ovation RNA-Seq system for Drosophila (NuGEN) . Standard protocol procedure with integrated DNase treatment was used to make the sequencing libraries. Fragmentation was performed using a Covaris E220 Focusedultrasonicator with the recommended settings for 200 base pair target length. The library quality was evaluated on a Fragment Analyser using the DNF-920 DNA reagent kit.
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