Human HEK293 and mouse J744A.1 cells were plated 24 h prior to addition of ASOs, which were dissolved in PBS at the indicated concentrations. After an ASO incubation time of 72 h, cells were lysed and total RNA was extracted using the PureLink Pro 96 RNA purification kit (Thermo Fisher Scientific), and quantitative reverse transcriptase PCR (qRT-PCR) for gene expression analysis was performed as described below. Primary mouse hepatocytes were isolated from C57BL/6 mice by a two-step collagenase liver perfusion method.52 (link) Freshly isolated hepatocytes were plated in collagen-l-coated 96-well plates at 25,000 cells/well in William’s medium E, supplemented with 1× penicillin (Pen)/streptomycin (Strep)/glutamine (Invitrogen) and 10% fetal bovine serum (ATCC). 3 h after plating, the hepatocytes were treated with ASOs at the indicated concentrations for 72 h, followed by RNA extraction and qRT-PCR as described below.
Purelink pro 96 rna purification kit
Manufactured by Thermo Fisher Scientific
The PureLink Pro 96 RNA purification kit is a high-throughput solution for extracting and purifying RNA from various sample types. It utilizes a silica-based membrane technology to efficiently capture and elute RNA molecules. The kit is designed to provide reliable and consistent RNA yields for downstream applications such as RT-qPCR, RNA sequencing, and microarray analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Sybr green 1 master mix, by Roche (1 mentions)
Hepes, by Merck Group (1 mentions)
Taqman reverse transcription reagent, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Cacl2, by Merck Group (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Hepg2 cell, by American Type Culture Collection (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Collagenase, by Worthington (1 mentions)
Hiseq platform, by Illumina (1 mentions)
5 mm stainless steel bead, by Qiagen (1 mentions)
Nanodrop 8000, by Thermo Fisher Scientific (1 mentions)
Purelink dnase, by Thermo Fisher Scientific (1 mentions)
4 protocols using purelink pro 96 rna purification kit
1
Quantitative analysis of gene expression in cell lines and primary hepatocytes
2
Atlantic Salmon Hepatocyte In Vitro Study
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Atlantic salmon (Salmo salar, 200 g) for the in vitro study were obtained from NIVA. HepG2 cells (derived from the liver tissue of a patient with hepatocellular carcinoma) were purchased from American Type Culture Collection. NaCl, KCl, HEPES, EDTA, CaCl2, L-15, fetal bovine serum (FBS), sodium bicarbonate, penicillin-streptomycin solution (100×), Trypan blue, bovine serum albumin (BSA), PBS, Tris, peroxidase, FAD, NaOH, 2'7'-dichlorofluorescin diacetate (reduced form), palmitoyl coenzyme A lithium salt (PalmCoA) and 18 : 3n-3 were purchased from Sigma-Aldrich. L-15 Glutamax was obtained from Invitrogen, metacain from Norsk Medisinaldepot, laminin and chloroform from Merck Millipore, collagenase from Worthington, 14 C-18 : 3n-3 from American Radiolabel Chemicals Inc., RNeasy Plus Mini Kit from Qiagen, Dulbecco's modified Eagle's medium (DMEM), PureLink Pro 96 RNA Purification Kit, PureLink Dnase set and primers from Thermo Fisher Scientific, SYBR Green I Master mix and LightCycler®480 from Roche Applied Science, TaqMan ® Reverse Transcription Reagents from Applied Biosystems, cetoleic acid (22 : 1n-11) from BOC Sciences and lactate dehydrogenase assay from Abcam. Ecoscint A scintillation liquid was purchased from National Diagnostics Inc. The scintillation counter TRI-CARB 1900 TR was obtained from Packard Instrument Co.
3
Transcriptome Analysis of Fish Tissues
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Total RNA was extracted from liver and skeletal muscle tissues of each individual fish using the PureLink Pro 96 RNA Purification Kit (Invitrogen), according to the manufacturer’s instruction. RNA was treated with PureLink On-Column DNase Digestion (Invitrogen) to remove any contaminating DNA. Samples were shipped to The Norwegian High-Throughput Sequencing Centre, where the mRNA library preparation and sequencing of transcripts were performed using standard protocols (www.illumina.com ). Samples were sequenced on an Illumina HiSeq platform as paired-end 151 bp reads. After final quality control, results from 48 liver- and 59 skeletal muscle samples remained for analyses.
Processing of reads, alignment and annotation was performed according to Moghadam et al.42 (link). Expression data were normalized via the median of the geometric means of fragment counts across all sample43 (link). Cufflinks and Cuffdiff were used to estimate the expression abundances of the assembled genes and transcripts44 (link).
Processing of reads, alignment and annotation was performed according to Moghadam et al.42 (link). Expression data were normalized via the median of the geometric means of fragment counts across all sample43 (link). Cufflinks and Cuffdiff were used to estimate the expression abundances of the assembled genes and transcripts44 (link).
4
RNA Extraction and QC for Ileum and Colon
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Frozen samples of ileum and colon tissues (~0.5 × 0.5 cm) were homogenized, and the RNA in the samples was precipitated as described in the procedural guidelines of TRIzol reagent (Ambion, Carlsbad, CA) using 5 mm stainless steel beads (Qiagen, Hilden, Germany). Total RNA was purified using PureLink Pro 96 RNA Purification Kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The RNA was treated on-column with PureLink DNase (Invitrogen) to remove residual genomic DNA. Concentrations of RNA were measured by using a NanoDrop TM 8000 spectrophotometer (Thermo Fisher Scientific, Wilmington), and the RNA integrity value (RIN) was assessed by using a 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). All samples had a RIN above 8 and they were sent for sequencing at the Norwegian Sequencing Centre (Oslo, Norway).
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