RNA of PMA-induced THP-1 cells treated with NC or ferroptosis-induced MDA-MB-231 cell derived-exosomes was extracted using the RNeasy min kit (Qiagen, Hilden, Germany). cDNA was synthesized by using the RT2 First Strand kit (Qiagen). Afterwards, the PCR reaction was performed as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15s and 60 °C for 1min. All samples were analyzed using the 2-ΔΔCt method with GAPDH as an internal control for normalization. The protein–protein network was constructed using Cytoscape V3.6.0.
Rneasy min kit
Manufactured by Qiagen
Sourced in United States
The RNeasy Mini Kit is a widely-used RNA extraction and purification system. It utilizes silica-membrane technology to efficiently isolate high-quality total RNA from a variety of sample types, including cells, tissues, and microorganisms. The kit provides a fast and reliable method for extracting total RNA suitable for downstream applications such as RT-PCR, Northern blotting, and microarray analysis.
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Lab products found in correlation
Nanodrop 2000c, by Thermo Fisher Scientific (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Iscript supermix kit, by Bio-Rad (2 mentions)
Tissuelyser, by Qiagen (2 mentions)
Truseq stranded total rna ribo zero kit, by Illumina (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Rt2 first strand kit, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
Sybr green master mix, by Bio-Rad (1 mentions)
Oligo dt beads, by Thermo Fisher Scientific (1 mentions)
Dl ap5, by Thermo Fisher Scientific (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Prism 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
High capacity reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Cfx96 thermocycler, by Bio-Rad (1 mentions)
Human β amyloid 40 elisa kit, by Fujifilm (1 mentions)
15 protocols using rneasy min kit
1
Exosome-induced Ferroptosis in THP-1 Cells
2
RNA Extraction from Cultured Cells
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Prepared cells were harvested and RNAs were isolated using TRIzol reagent (Ambion, Carlsbad, CA, USA) or RNeasy min kit (Qiagen, Germantown, MD, USA). The purity and integrity of total RNA were checked using NanoDrop One (Thermo Scientific, Waltham, MA, USA).
3
Encapsidated pgRNA Extraction Protocol
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Cells from one well of a six-well plate were washed with PBS and incubated with 500 μL lysis buffer [50 mM Tris⋅HCl (pH7.5), 1 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, and 1% Nonidet P-40 with Protease inhibitor mixture] in 37 °C for 10 min. The lysates were centrifuged for 2 min at 14,000 rpm to remove the cell debris and nuclei. The supernatant was transferred to a new centrifuge tube and then incubated with 6 U micrococcal nuclease (New England Biolabs) and 30 μL of 100 mM CaCl2 for 15 min at 37 °C for to remove unprotected free nucleic acids. The encapsidated pgRNA was extracted by the RNeasy min kit (Qiagen). The extracted encapsidated pgRNA was analyzed with RT-qPCR and Northern blot assay.
4
RNA Isolation and qPCR Analysis Protocol
5
Neuronal RNA Isolation and Enrichment
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After being cultured in vitro for 6 days, neuronal cells were silenced with 1 μM tetrodotoxin (TTX; Fisher, Clarion County, PA, USA) and 100 μM DL-2-amino-5-phosphopentanoic acid (DL-AP5; Fisher) overnight. The next morning, neuronal cells were depolarized with 55 mM KCl for 0 h, 2 h, and 6 h. At the end time point, the neuronal cells were harvested and lysed with TRIzol reagent. Total RNA was extracted using TRIzol reagent combined with the RNeasy min kit (QIAGEN, Germantown, MD, USA) with DNase I on-column digestion. To enrich poly(A)-containing mRNAs, two rounds of poly(A) selection were performed using oligo(dT) beads (Thermo Fisher) following the manufacturer’s instructions.
6
Transcriptomic Analysis of Senescent Cell Reversion
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RNA samples were prepared by RNeasy min kit (Qiagen); a mixture of three biological RNA replicates was used to represent each condition (parental, senescent, revertant). RNA-seq analysis, including rRNA depletion, library preparation, multiplexing and cluster generation, sequencing on Illumina HiSeq2500, and differential gene expression analysis, was performed by Genewiz (South Plainfield, NJ, USA). Gene function analysis was performed using DAVID (Database for Annotation, Visualization and Integrated Discovery, https://david.ncifcrf.gov/ ) and PANTHER (Protein Analysis THrough Evolutionary Relationships, http://pantherdb.org ) programs.
7
RNA Extraction and qRT-PCR Analysis
8
RNA Extraction and Real-Time qPCR Analysis
9
Nematode RNA Isolation and Gene Expression Analysis
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RNA was isolated from between 1000 and 2000 nematodes according to the Qiagen RNeasy Min Kit protocol. cDNA was created from 100 ng of the isolated RNA using the High Capacity Reverse Transcription kit (Life Technologies) per the manufacturer's instructions. Gene expression was measured via real-time PCR using the Power SYBR Green PCR Master Mix (Life Technologies). Changes in expression levels were calculated based on the standard delta-delta-cT method, compared to housekeeping genes cdc-42 and pmp-3. Primer sequences and PCR conditions can be found in Table S2 .
10
Cardiac Gene Expression Profiling in Mice
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Frozen heart tissues of 3‐month‐old mice were used for the isolation of total RNA. Tissues were homogenized with a TissueLyser (Qiagen, Boston, MA) using Trizol buffer followed by transferring to a chloroform containing tube and centrifuge. The supernatant was mixed with equal volume of 70% ethanol and loaded onto a Qiagen RNA processing column from the Qiagen RNeasy Min‐Kit (Qiagen, Boston, MA). All steps were followed according to the manufacturer's instructions. RNA quality and concentration was determined via NanoDrop 2000c (ThermoScientific, Wilmington, DE). A known amount of RNA was reverse transcribed to generate cDNA using the iScript Supermix kit (Bio‐Rad, Hercules, CA) on a CFX96 Thermocycler (BioRad, Hercules, CA). Primers were used at a final concentration of 0.25 to 0.5 μmol/L for target genes and normalized to GAPDH expression. Relative gene expression levels were quantified using the formula 2−ΔΔCt. A 3‐step amplification protocol was implemented: 10 minutes of denaturation at 95°C, followed by 45 cycles of denaturation (95°C, 1 second), annealing (65°C, 10 seconds), and extension (72°C, 20 seconds). Gene‐specific primer sequences are presented in Table 1 .
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