For performing the microarray analysis, 3 paired of tumor tissues and nontumor tissues were obtained from HCC patients. Then total RNAs were collected from them and were transferred into the fluorescent cRNA by using an Arraystar Super RNA Labeling Kit (Arraystar, USA). Moreover, they were purified by RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Additionally, the microarray hybridizations on Arraystar Human circRNA Array chip (Agilent, USA) were performed according to its protocol. Finally, it was scanned by an Agilent Scanner (Agilent, Santa Clara, CA, USA), which was analyzed with the Agilent supporting software.
Super rna labeling kit
Manufactured by Arraystar
Sourced in United States, China
The Arraystar Super RNA Labeling Kit is a lab equipment product designed for labeling RNA samples. It provides a simple and efficient method to label RNA with fluorescent dyes for use in various applications, such as microarray analysis and RNA-sequencing. The kit includes all necessary reagents and components to facilitate the labeling process.
Automatically generated - may contain errors
Lab products found in correlation
Agilent scanner g2505c, by Agilent Technologies (120 mentions)
Rnase r, by Illumina (115 mentions)
Human circrna array v2, by Arraystar (49 mentions)
Rneasy mini kit, by Qiagen (46 mentions)
Scanner g2505c, by Agilent Technologies (28 mentions)
Human circrna array, by Arraystar (24 mentions)
Trizol reagent, by Thermo Fisher Scientific (23 mentions)
Hybridization oven, by Agilent Technologies (21 mentions)
Agilent feature extraction software, by Agilent Technologies (19 mentions)
Agilent hybridization oven, by Agilent Technologies (15 mentions)
Agilent feature extraction software version 11.0.1.1, by Agilent Technologies (14 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (14 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (14 mentions)
Feature extraction software, by Agilent Technologies (11 mentions)
Mouse circrna array v2, by Arraystar (9 mentions)
Rnase r epicentre, by Illumina (6 mentions)
Rat circrna array, by Arraystar (5 mentions)
Agilent microarray scanner, by Agilent Technologies (5 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Agilent feature extraction software version 11.0, by Agilent Technologies (4 mentions)
194 protocols using super rna labeling kit
1
Microarray Analysis of HCC Tissues
2
CircRNA Profiling in Cancer Samples
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After being obtained from surgical specimens, samples were immediately frozen using liquid nitrogen. Sample preparation and microarray hybridization were performed according to the protocols of Arraystar (Rockville, MD, USA). CircRNAs were enriched through removing linear RNAs with Rnase R (Epicentre, Madison, WI, USA), and then amplified and labelled using Arraystar Super RNA Labeling Kit (Arraystar). mRNAs were purified using rRNA removal kit (Arraystar). Subsequently, Arraystar Human circRNA Array (8 × 15K) and LncPath Human Cancer Array were used for hybridization, and then scanned by the Agilent Scanner G2505C (Jamul, CA, USA). circRNAs and mRNAs demonstrating fold-changes of ≥ 2 and p-values of less than 0.05 were regarded as significantly differentially expressed.
3
Circular RNA Profiling from Total RNA
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Total RNA from each sample was quantified using a NanoDrop ND-1000. The sample preparation and microarray hybridization were performed based on Arraystar's standard protocols (KANGCHEN Biotech, Shanghai, China). In brief, total RNA was digested with RNase R (Epicentre, Inc.) to remove linear RNAs and enrich for circular RNAs. Then, the enriched circRNAs were amplified and transcribed into fluorescent cRNA using a random priming method (Arraystar Super RNA Labeling Kit; Arraystar). The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array V2 (8×15K, Arraystar). Next, the slides were washed and the arrays were scanned using an Agilent Scanner G2505C. Agilent Feature Extraction software (version 11.0.1.1) was applied to analyze the acquired array images. Quantile normalization and subsequent data processing were performed using the R software limma package. Differentially expressed circRNAs with statistical significance between two groups were identified through Volcano Plot filtering. Significantly differentially expressed circRNAs were then further filtered through analysis of Fold Change. Hierarchical Clustering was performed to highlight distinguishable circRNAs expression patterns among samples.
4
Circular RNA Expression Microarray Analysis
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The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array V2 (8×15K, Arraystar, USA). Sample labeling and array hybridization were performed according to the manufacturer’s instructions (Arraystar, USA). Briefly, enriched circRNAs were transcribed into fluorescent-labeled cRNAs by an Arraystar Super RNA Labeling Kit (Arraystar, USA) and then purified using an RNeasy Mini Kit (Qiagen, USA). A total of 1 μg of cRNA from each sample was fragmented by blocking agent and fragmentation buffer and incubated at 60°C for 30 min. Hybridization buffer was used to dilute the labeled cRNA. Then, the mixture was loaded onto a circRNA expression microarray slide. The slides were incubated at 65°C for 17 h in an Agilent Hybridization Oven (Agilent Technologies, USA). Next, the slides were washed, fixed, and scanned by Agilent Scanner G2505C (Agilent Technologies, USA).
5
Profiling Circular RNAs in Cell Lines
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The panel of cell lines (three biological replicates for each) were profiled using the Arraystar Human circRNA Array version 2.0 (Arraystar, MD, United States). The sample preparation and microarray hybridization were performed according to manufacturer’s instructions. Briefly, total RNA was digested with RNAse R (Epicenter, Illumina, CA, United States) to remove linear RNAs and enrich for circRNAs. The enriched circRNAs were amplified and transcribed into fluorescent cRNA using a random priming method with Arraystar Super RNA Labeling Kit (Arraystar). The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array V2 (8 × 15 K). The array slides were washed and scanned on the Agilent Scanner G2505C. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images.
6
Rat circRNA Microarray Analysis Protocol
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Sample preparation and microarray hybridization were performed based on Arraystar's standard protocols. Briefly, total RNA was digested with RNase R (Epicenter, Inc.) to remove linear RNAs and enrich circRNAs. Then, the enriched circRNAs were amplified and transcribed into fluorescent cRNA using a random priming method (Arraystar Super RNA Labeling Kit; Arraystar). The labeled cRNAs were hybridized onto the Arraystar Rat circRNA Array (8x15K, Arraystar) and incubated for 17 h at 65°C in an Aligent Hybridization Oven. After the slides were washed, the arrays were scanned by an Agilent G2505C scanner.
7
Circulating RNA Profiling by Microarray
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Fluorescent cRNAs obtained from three pairs of tissue samples were labeled by the random labeling method with the Arraystar Super RNA Labeling Kit (Arraystar, USA). The labeled circRNA hybridized to the Arraystar Human circRNA Array V2 (8x15K, Arraystar). The microarray was scanned with Agilent Scanner G2505C. Image analysis was conducted using Agilent Feature Extraction software (version 11.0.1.1). circRNA data were analyzed using the GeneSPring 13.0 software (Agilent). Differentially expressed circRNAs were those with fold change>2.0 and P<0.05.
8
Circular RNA Expression Profiling
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Sample labeling and array hybridization were performed according to the manufacturers' protocols as described below. To enrich circRNAs, linear RNAs were removed using Rnase R (Epicentre; Illumina, Inc.) to digest total RNAs. Each sample of enriched circRNAs was then amplified and transcribed into fluorescent cRNA using the treating random primers method (Arraystar Super RNA Labeling kit; Arraystar, Inc.). The labeled cRNAs were purified using the RNeasy Mini kit (Qiagen, Inc.). The concentration and specific activity of the labeled cRNAs (pmol Cy3/µg cRNA) were measured using a NanoDrop ND-1000. A total of 1 µg of each labeled cRNA was fragmented by adding 5 µl 10X Blocking Agent (Agilent Technologies, Inc.) and 1 µl of 25X Fragmentation Buffer (Agilent Technologies, Inc.), and the mixture was incubated at 60°C for 30 min. A total of 25 µl 2X Hybridization buffer (Agilent Technologies, Inc.) was added to dilute the labeled cRNA, 50 µl of hybridization solution was added into the gasket slide and assembled with the circRNA expression microarray slide. The slides were then incubated for 17 h at 65°C in an Agilent Hybridization Oven (G2545A; Agilent Technologies, Inc.). Finally, following washing and fixing the slides, the hybridized arrays were scanned using the Agilent Scanner G2505C (Agilent Technologies, Inc.).
9
Profiling Circular RNA Transcriptome in Disease
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Microarray analysis of circRNA expression was performed on a subset of 20 samples—12 patients (6 females, 6 males) and 8 age- and sex-matched controls. Total RNA from each sample was prepared for the microarray analysis according to the manufacturer’s protocol (Arraystar, USA). Briefly, total RNA was digested with RNase R (Epicentre, Inc., USA) to enrich circular RNAs. Enriched circular RNAs were amplified and transcribed into fluorescent complimentary RNA utilizing a random priming method (Arraystar Super RNA Labeling Kit; Arraystar, USA) and then hybridized onto the Arraystar Human circRNA Array V2 (8x15K, Arraystar, USA). Slides were washed and the arrays were afterwards scanned by the Agilent Scanner G2505C.
Acquired array images were analyzed using Agilent Feature Extraction software (version 11.0.1.1). Quantile normalization and subsequent data processing were performed using the R software limma package. Differentially expressed circRNAs with statistical significance between two groups were identified through volcano plot filtering. Fold change filtering was used to identify differentially expressed circRNAs between two samples. Distinguishable circRNA expression patterns among samples were identified through hierarchical clustering.
Acquired array images were analyzed using Agilent Feature Extraction software (version 11.0.1.1). Quantile normalization and subsequent data processing were performed using the R software limma package. Differentially expressed circRNAs with statistical significance between two groups were identified through volcano plot filtering. Fold change filtering was used to identify differentially expressed circRNAs between two samples. Distinguishable circRNA expression patterns among samples were identified through hierarchical clustering.
10
Circular RNA Enrichment and Microarray
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Having homogenized with TRIzol reagent (Invitrogen, Waltham, MA, USA) by the Mini-Bead-Beater-16 (Biospec, United States), Total RNA of three pairs of collected samples were then extracted used RNeasy mini-kit (Qiagen, Hilden, Germany), while simultaneous digestion was performed with DNase (Baseline Zero DNase, Epicentral, USA). NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to evaluate RNA quantity, and denaturing agarose gel electrophoresis was applied to assessed RNA integrity, as we described before (Jiang et al., 2020 (link)). After the required RNA samples were prepared, microarray hybridization was performed according to the standard protocol of ArrayStar. For enriching circular RNAs, Rnase R (Epicentre, Inc.) was used in total RNAs to remove linear RNAs. Then a random priming method (Arraystar Super RNA Labeling Kit; Arraystar) was utilized to amplify and transcribe the enriched circular RNAs into fluorescent cRNA. The fluorescent cRNAs were hybridized by the Arraystar Human circRNA Array V2 (8x15K, Arraystar). The Agilent Scanner G2505C was used to scan arrays after the slides having been washed. Acquired array images was analyzed by Agilent Feature Extraction software (version 11.0.1.1) (Zou et al., 2020 (link)). R software limma package was used for quantile normalization and subsequent data processing.
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