Total RNA was extracted from MCF7 and MCF10A cells in both 2D and 3D cultures using RNeasy plus kits (Qiagen, Germany) according to manufacturer’s instructions. Briefly, cells grown as monolayer were trypsinized and washed with PBS twice followed by RNA extraction using RNeasy Plus Kit. To collect spheroids, medium was removed from 3D cell culture followed by washing with PBS twice gently. Pre-chilled cell recovery solution (Corning, USA) at the volume of 200 μl were added to 8-well chamber slide (Ibidi, Germany). Matrigel matrix was fully depolymerized, and spheroids were released after 30 min incubation at 4 °C. Suspended spheroids were collected and washed with PBS twice followed by RNA extraction using RNeasy Plus Kit (Qiagen). The integrity and quantity of RNA were determined with NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) before sending out for RNA sequencing.
Rneasy plus kit
Manufactured by Qiagen
Sourced in Germany, United States, United Kingdom, Japan, Australia, Canada, Switzerland, Spain, Sweden, Netherlands, France
The RNeasy Plus kit is a product designed for the purification of total RNA from a variety of sample types. It utilizes a silica-membrane-based technology to efficiently capture and purify RNA molecules.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (79 mentions)
Nanodrop, by Thermo Fisher Scientific (48 mentions)
Iscript cdna synthesis kit, by Bio-Rad (48 mentions)
Trizol, by Thermo Fisher Scientific (43 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (40 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (34 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (29 mentions)
Trizol reagent, by Thermo Fisher Scientific (26 mentions)
Superscript 3, by Thermo Fisher Scientific (25 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (25 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (25 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (23 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (22 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (22 mentions)
Quantitect reverse transcription kit, by Qiagen (18 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (18 mentions)
Hiseq 2500, by Illumina (17 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (17 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (16 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (15 mentions)
1 019 protocols using rneasy plus kit
1
RNA Extraction from 2D and 3D Cell Cultures
2
RNA Extraction and ToBRFV Detection
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One hundred milligrams of leaf sample was collected and homogenized in 600 μl of Buffer RLT Plus, with 1% β-mercaptoethanol (RNeasy Plus kit, Qiagen, USA) using Bead Ruptor 24 (Omni International, Inc.) at 5.5 m/s for two cycles of 20 s with a dwelling step of 30 s between cycles, and PowerBead Tubes with 1.4-mm ceramic beads (Qiagen, USA). RNA was purified using the RNeasy Plus kit (Qiagen, USA).
Detection of ToBRFV was performed with the ISF-ISHI-Veg RT-qPCR protocol (ISF-ISHI-Veg, 2020 ).
Detection of ToBRFV was performed with the ISF-ISHI-Veg RT-qPCR protocol (ISF-ISHI-Veg, 2020 ).
3
RNA Extraction and Quantification
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Total RNA was isolated employing the RNeasy Plus kit (Qiagen) according to manufacturer's protocol. Extracted RNA was reversed transcribed into cDNA using random hexamer priming and Superscript III (ThermoFisher) according to the protocol supplied by the manufacturer. For qPCR, cDNA templates were amplified, and the CT values were quantified with PowerUp SYBR Green Master Mix (ThermoFisher), and normalized to actin as an internal control. Experiments were performed using a StepOnePlus Real-Time PCR System (Applied Biosystems). poly(A)-mRNA was isolated using Ambion® Poly(A)Purist™ MAG Kit (ThermoFisher) according to manufacturer's protocol. Nuclear and cytoplasmic separation of RNA was performed with Thermo Scientific NE PER Nuclear and Cytoplasmic Extraction Kit (ThermoFisher) according to manufacturer's protocol. RNA in either the nuclear pellets or cytoplasmic fractions was extracted with RNeasy Plus kit (Qiagen). Total RNA extracted per fraction was quantified and used to calculate the percentage of mRNA localization for the RT-qPCR quantification. The list of primers is provided in Supplementary Data (Supplementary Table S1 ).
4
Spleen RNA Extraction Protocol
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RNA extraction was performed on the spleens of mice (n = 2–3) which had been infected 8 weeks previously using the RNeasy Plus Kit (Qiagen, Hilden, Germany). The procedure included a DNA removal step according to the manufacturer’s protocol. Before the protocol was applied, the spleen in its entirety was homogenized in liquid nitrogen using a cryo mortar and a pestle with the addition of 1ml chaotropic buffer (RLT Plus) from the RNeasy Plus Kit (Qiagen, Hilden, Germany). The tissue/buffer powder was transferred to a 1.5-ml reaction tube and, after thawing, an aliquot of 50 μl was taken and diluted with 550 μl RNeasy Plus buffer for the cleanup process. Finally, a phenol/chloroform extraction (phenol/chloroform/IAA, 25:24:1, pH 6.6; Thermo Fisher Scientific, Waltham, MA, USA) was performed. This procedure enabled us toaverage the expression pattern of all spleen substructures and ensure that we did not overload the RNeasy spin column. The whole RNA samples were quantified spectrophotometrically (Nanodrop 1000, Thermo Fisher Scientific) and diluted to a concentration of 70 ng/μl. RNA integrity was tested using an Agilent RNA 6000 Nano Chip with a Bioanalyzer 2100 instrument (Agilent Technologies). The samples achieved RNA integrity numbers between 8.6 and 9.8.
5
RNA Isolation from Cell Cultures
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Cells were seeded in six-well TC-treated plates in triplicate. The following day, treatments were applied, and cells were incubated as indicated at 37°C in Assay Media with 10% FBS. Following incubation, media was aspirated fully and 0.6 mL RLT buffer with β-mercaptoethanol (from Qiagen RNeasy Plus kit) was added to wells and plates were frozen at −20°C. Plates were thawed on ice, wells were scraped, and contents transferred to QIAshredder columns. RNA was isolated according to the RNeasy Plus kit instructions (Qiagen). RNA was eluted, quantified, aliquoted for RNA-seq submission and/or qPCR, then frozen at −80°C.
6
RNA Extraction from Yeast and Bacteria
7
Transcriptomic Analysis of DAB2IP Knockdown
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DLD-1 cells were infected with FB LACZ or FB DAB2IP and subsequently selected in neomycin. 7 days later, 5×105 cells were plated in triplicate in 6-well plates. RNA was harvested 2 days later using RNeasy Plus kit (Qiagen, catalog no. 74134). RNA was sequenced at Dana-Farber Cancer Institute Molecular Biology Core Facility using the Illumina NextSeq500. Raw data was mapped to the Hg19 genome using STAR (RRID:SCR_004463) and count files were made using HTSeq48,49 (GEO ID GSE224869). HCT116 cells were infected with shCNT or shDAB2IP and subsequently selected with puromycin. Seven days later, 5×105 cells were plated in triplicate in 6-well plates. RNA was harvested 2 days later using RNeasy Plus kit (Qiagen, catalog no. 74134). RNA was sequenced by Novogene (GEO ID GSE224870).
8
Quantitative RT-PCR of Pten Gene Expression
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Total RNA was extracted from Pten+/+ and PtenY68H/+ flash frozen cortex using the RNeasy Plus kit (RNeasy Plus kit, # 74136, Qiagen, Germantown, MD) and cDNA synthesis using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Maxima First Strand cDNA Synthesis Kit for RT-qPCR, #K1642, Thermo-Fisher) according to supplier protocols. We designed primers using UCSC Genome browser mouse GRCm39/mm39 mouse (https://genome.ucsc.edu/ ) assembly to select exonic regions of interest and selected primers according to standard Primer3 version 0.4.0 (https://bioinfo.ut.ee/primer3-0.4.0/ ) protocol to select our qRT-PCR primers. qRT-PCR was performed using Power SYBR Green Master Mix (SYBR Green Master Mix, # 4367660, Thermo-Fisher) following standard protocol to prepare samples in a 96-well plate (Fisherbrand 96-Well Semi-Skirted PCR Plates, #14230244, Fisher Scientific, Waltham, MA) and run on QuantStudio™ 3 Real-Time PCR System (QuantStudio™ 3 Real-Time PCR System, # A28567, Thermo-Fisher) as a standard run with cycling program of 10 min at 95 °C followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Primer sequences used and designed for mouse are listed in Additional file 1 : Table S1.
9
Comprehensive Transcriptome Analysis Protocol
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Total RNA was isolated employing the RNeasy Plus kit (Qiagen) according to manufacturer's protocol.
Extracted RNA was reversed transcribed into cDNA using random hexamer priming and Superscript III (ThermoFisher) according to the protocol supplied by the manufacturer. For qPCR, cDNA templates were amplified, and the C T values were quantified with PowerUp SYBR Green Master Mix (ThermoFisher), and normalized to actin as an internal control. Experiments were performed using a
StepOnePlus Real-Time PCR System (Applied Biosystems). poly(A)-mRNA was isolated using Ambion® Poly(A)Purist™ MAG Kit (ThermoFisher) according to manufacturer's protocol. Nuclear and cytoplasmic separation of RNA was performed with Thermo Scientific NE PER Nuclear and Cytoplasmic Extraction Kit (ThermoFisher) according to manufacturer's protocol. RNA in either the nuclear pellets or cytoplasmic fractions was extracted with RNeasy Plus kit (Qiagen). Total RNA extracted per fraction was quantified and used to calculate the percentage of mRNA localization for the RT-qPCR quantification. The list of primers is provided in Supplementary Data (Supplementary Table S1).
Extracted RNA was reversed transcribed into cDNA using random hexamer priming and Superscript III (ThermoFisher) according to the protocol supplied by the manufacturer. For qPCR, cDNA templates were amplified, and the C T values were quantified with PowerUp SYBR Green Master Mix (ThermoFisher), and normalized to actin as an internal control. Experiments were performed using a
StepOnePlus Real-Time PCR System (Applied Biosystems). poly(A)-mRNA was isolated using Ambion® Poly(A)Purist™ MAG Kit (ThermoFisher) according to manufacturer's protocol. Nuclear and cytoplasmic separation of RNA was performed with Thermo Scientific NE PER Nuclear and Cytoplasmic Extraction Kit (ThermoFisher) according to manufacturer's protocol. RNA in either the nuclear pellets or cytoplasmic fractions was extracted with RNeasy Plus kit (Qiagen). Total RNA extracted per fraction was quantified and used to calculate the percentage of mRNA localization for the RT-qPCR quantification. The list of primers is provided in Supplementary Data (Supplementary Table S1).
10
Transcriptome Analysis of SHMT2 Knockout
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RNA was isolated from cell lines using RNeasy Plus kit (Quiagen) according to the manufacturers´ recommendation. Following the depletion of ribosomal RNA libraries were prepared according to the TruSeq Stranded Total RNA protocol (Illumina) and sequencing was performed on a HiSeq 2500 (Illumina). Analysis was performed using the Galaxy system45 (link) and the R software package46 . Adapter sequences were trimmed using Cutadapt (Galaxy version 1.6)47 and the trimmed reads were then mapped with TopHat (Galaxy Version 0.9)48 (link) to the GRCh38 reference using ENSEMBL version 80 genes as known splice junctions. The read counts per gene were determined using htseq-count (Galaxy Version 0.6.1galaxy1)49 (link) in ‘union’ mode. Differential expression analysis was performed in R using DESeq2 1.12.3 package50 (link). SHMT2 knockout gene expression (log2 reads per kilobase of transcript per million mapped reads (RPKM)) was graphed relative to the wild-type and the re-expressed cell lines.
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