For microarray analysis 1 µg of total RNA was labeled using the Flashtag RNA labeling kit for Affymetrix (Genisphere LLC., Hatfield, PA, USA) according to the manufacturer’s instructions. The labeled samples were hybridized to GeneChip miRNA Array (Affymetrix, Santa Clara, CA, USA). The Affymetrix miRNA array assay miRNAs includes small nucleolar RNAs (snoRNAs) and small Cajal Body specific RNAs (scaRNAs) in human. The 847 human miRNAs on the array are derived from Sanger miRbase miRNA database V11. Four copies of each miRNA probe are distributed on the array.
Flashtag rna labeling kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Flashtag RNA labeling kit is a tool designed for the detection and analysis of RNA molecules. It provides a simple and efficient method to label RNA for various applications, such as Northern blotting, in situ hybridization, and microarray analysis.
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Lab products found in correlation
Genechip mirna array, by Thermo Fisher Scientific (1 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (1 mentions)
Genechip mirna array 3, by Thermo Fisher Scientific (1 mentions)
Genechip scanner 3000 7g, by Thermo Fisher Scientific (1 mentions)
Mirna qc tool software, by Thermo Fisher Scientific (1 mentions)
Gene array 3000 scanner, by Thermo Fisher Scientific (1 mentions)
Fluidics station 450, by Thermo Fisher Scientific (1 mentions)
Genechip mirna 3.0 array, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
4 protocols using flashtag rna labeling kit
1
Microarray Analysis of Human miRNAs
2
Affymetrix MicroRNA Array Profiling
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MiRNA microarray analysis was performed using the Affymetrix Solution GeneChip MicroRNA Version 1.0 array (comprising 46228 probes with 7815 probes sets) according to manufacturer's (Affymetrix) instructions. Briefly, a starting material of 1 µg of small RNA was used for poly A tailing on the 3′ end of the small RNA followed by ligation of the biotinylated signal molecule to the small RNA samples, according to Affymetrix Flashtag RNA labeling kit (Affymetrix, p/N FT10AFYB). Hybridzation procedure was performed as described in the manufacturer's protocol. The chips were scanned using Affymetrix Genechip Scanner 3000 and the data was extracted using Genechip Operating Software (GCOS).
The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE54439.
The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE54439.
3
miRNA Expression Profiling with GeneChip
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Total RNA including low molecular weight RNA was labeled using the Flashtag RNA labeling kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. Briefly, for each sample, 400 ng of total RNA was subjected to a tailing reaction followed by the ligation of the biotinylated signal molecule to the target RNA sample. Each sample was hybridized to a GeneChip® miRNA Array 3.0 (Affymetrix, Santa Clara, CA, USA) for 16 h at 48 °C at 60 rpm. After washing and staining with a Fluidics Station 450 (Fluidics script FS450_0002), arrays were scanned with a GeneChip® Scanner 3000 7G (Affymetrix). The image data were analyzed with the Expression Console Software (www.affymetrix.com ) for quality control. GeneChip® miRNA Array 3.0 contained 19,724 total mature miRNA probe sets and covered 153 organisms. This array contained 1111 and 855 mouse mature miRNA and pre-miRNA, respectively. miRNA probes of this array were derived from the Sanger miRBase miRNA database v17.
4
Exosomal miRNA Profiling and Analysis
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RNA was isolated from different exosomes and cell preparations using total exosomal RNA, and a miRNA isolation kits in accordance with the manufacturer's instructions (Fig. 12). The RNA was quantified spectrophotometrically and its quality was analyzed using an Agilent 2100 Bioanalyzer. To characterize the miRNA expression profiles of exosomes and exosome-treated cells and to detect differences in the expression profiles of the cells, a miRNA microarray was performed. In this sense, miRNAs from K562S, K562R, Sexo, Rexo, and K562S + Rexo were labeled using an exosomal RNA Flash Tag RNA labeling kit in accordance with the recommendations of the Affymetrix manufacturer. Next, cells were washed under standard conditions using an Affymetrix Fluidics Station 450 and a hybridization oven 640. Affymetrix Gene Array 3000 scanner was used to process the images, and Affymetrix miRNA QC Tool software was used to analyze the raw data files of the samples. The Affymetrix Gene Chip miRNA 3.0 Array included 19,724 probes and 1733 human adult miRNAs (Fig. 12). Pairwise comparisons of groups were made to exclude miRNAs with less than two fold changes.
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