Total RNA was extracted from the tumor tissues and normal tissues from ten gastric cancer patients (including 5 node-negative and 5 node-positive patients) and was prepared using mirVana miRNA Isolation kit (Invitrogen), according to the manufacturer's instruction. RNA concentration and purity were assessed by spectrophotometric analysis. The quality of small RNAs in each sample was determined using the 2100 Bioanalyzer assay (Agilent Technologies, Santa Clara, CA, USA). MiRNA microarray experiments were carried out by using the Agilent Human miRNA Microarray Kit version 3 (Based on Sanger miRbase release 12.0) with probe sets for 470 human miRNAs (miRBase release 9.1). For each sample, 100 ng total RNA was hybridized with the miRNA array and further processed according to Agilent's miRNA Microarray System protocol. The arrays were scanned with an Agilent Technology G2565B scanner (Agilent Technologies). The scanned images were gridded and analyzed by using Agilent Feature Extraction Software version 10.1.
2100 bioanalyzer assay
Manufactured by Agilent Technologies
Sourced in United States
The 2100 Bioanalyzer is a lab instrument for automated electrophoretic separation and analysis of DNA, RNA, proteins, and cells. It provides quantitative and qualitative assessment of these biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Mirna microarray system protocol, by Agilent Technologies (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Agilent surescan microarray scanner, by Agilent Technologies (1 mentions)
Mouse mirna microarray, by Agilent Technologies (1 mentions)
Mirna microarray system with mirna complete labeling and hyb kit, by Agilent Technologies (1 mentions)
Rq1 dnase, by Promega (1 mentions)
Feature extraction software, by Agilent Technologies (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
4 protocols using 2100 bioanalyzer assay
1
Gastric Cancer miRNA Profiling Protocol
2
Exosomal miRNA Profiling by Microarray
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Exosome-RNA extraction was using a miRNeasy kits (Qiagen, Hilden, Germany), according to the manufacturer’s instruction. RNA concentration and purity were assessed by spectrophotometric analysis. The quality of small RNAs in each sample was determined using the 2100 Bioanalyzer assay (Agilent Technologies, Santa Clara, CA, USA). MiRNA microarray experiments were performed by using the Agilent Human miRNA Microarray Kit version 3 with probe sets for 470 human miRNAs. For each sample, 100 ng total RNA was hybridized with the miRNA array and further processed according to Agilent’s miRNA Microarray System protocol. The scanned images were gridded and analyzed by using Agilent Feature Extraction Software version 10.1.
3
Profiling miRNA Expression in Treatment Outcomes
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Blood was collected before treatment in PAXgene and total RNAs, including small RNAs, were extracted as previously described (22 (link)). RNA quality was assessed using the 2100 Bioanalyzer assay (Agilent, Les Ulis, France), and according to the criteria of the Minimum Information for publication of Quantitative real-time PCR Experiments MIQE guidelines, only samples with a RIN>8 were used. Sixteen patients were analyzed with the TaqMan Low-Density Array (TLDA) technology as previously described (22 (link)) and divided in two groups according to treatment response outcome. One group was composed of 8 patients with CR with blood and BM uMRD. The other group was composed of 8 patients with no-CR and detectable MRD. Validation of miRNA expression levels were quantified in a second cohort of 100 treatment-naive patients using multiplex Taqman microRNA assays (Life Technologies, Saint Aubin, France) as previously described (22 (link)).
4
Mouse Osteoclast miRNA Profiling
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Primary osteoclast precursors were plated at 500,000 cells/well in 6 well plates. Total RNA was isolated from differentiating cultures using the miRNeasy Mini kit (Qiagen, Valencia, CA). On-column DNase treatment was performed to reduce contamination with genomic DNA, and an additional treatment with RQ1 DNase (Promega, Madison, WI) was performed prior to gene expression analysis. RNA concentration and purity were assessed by spectrophotometric analysis. The quality of small RNAs in each sample was determined using the 2100 Bioanalyzer assay (Agilent Technologies, Santa Clara, CA).
Four independent cultures were analyzed for each time point. For each sample, 200 ng of total RNA were labeled using miRNA Microarray System with miRNA Complete Labeling and Hyb Kit (Agilent Technologies). According to the manufacturer’s instructions, the samples were hybridized for 20 hours onto a mouse miRNA Microarray, Release 15.0, 8×15 K (based on Sanger miRBase release 15.0), containing 627 mouse mature miRNA probes (Agilent Technologies). Hybridized and washed array slides were scanned at 5 µm resolution using an Agilent SureScan Microarray Scanner (Agilent Technologies). Image processing was completed using Feature Extraction Software (Agilent Technologies).
Four independent cultures were analyzed for each time point. For each sample, 200 ng of total RNA were labeled using miRNA Microarray System with miRNA Complete Labeling and Hyb Kit (Agilent Technologies). According to the manufacturer’s instructions, the samples were hybridized for 20 hours onto a mouse miRNA Microarray, Release 15.0, 8×15 K (based on Sanger miRBase release 15.0), containing 627 mouse mature miRNA probes (Agilent Technologies). Hybridized and washed array slides were scanned at 5 µm resolution using an Agilent SureScan Microarray Scanner (Agilent Technologies). Image processing was completed using Feature Extraction Software (Agilent Technologies).
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