T4 rna ligase

Manufactured by GE Healthcare

Sourced in United Kingdom

T4 RNA ligase is an enzyme commonly used in molecular biology laboratories. It catalyzes the formation of a phosphodiester bond between the 5' phosphate and 3' hydroxyl groups of RNA molecules. This enzyme is essential for various RNA-related applications, including RNA library preparation, RNA sequencing, and RNA structure analysis.

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5 protocols using t4 rna ligase

Identification of Differentially Expressed miRNAs in EGR1 Treatment

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The microarray analysis was conducted to identify differentially expressed miRNAs after EGR1 treatment. The TRIzol Reagent was used to extract total RNA. The RNA (100 ng) was dephosphorylated by calf intestinal alkaline phosphatase (GE Healthcare Europe GmbH, Penzberg, Germany) and denaturalized in dimethyl sulfoxide. The RNA was further labeled with pCp-Cy3 using a T4 RNA ligase (GE Healthcare). The labeled RNA was hybridized with Aglient miRNA microarray (Agilent Technologies, Palo Alto, CA, USA) for 20 h. Then, the microarrays were scanned using an Agilent SureScan Microarray scanner (Agilent). The data were extracted using an Agilent feature extraction 10.0 (Aglient) and the corresponding heatmaps were produced.

Zhang L., Ren R., Yang X., Ge Y., Zhang X, & Yuan H. (2021). Oncogenic role of early growth response-1 in liver cancer through the regulation of the microRNA-675/sestrin 3 and the Wnt/β-catenin signaling pathway. Bioengineered, 12(1), 5305-5322.

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miRNA Microarray Analysis of GSCs

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The SurePrint G3 Human miRNA 8x60K Microarray (G4872A, Agilent Technologies) was used according to the manufacturer's protocols. Total RNA was isolated from GSCs with TRIzol reagent (Life Technologies). In total, 100 ng of total RNA was labelled with pCp-Cy3 (Agilent Technologies) and 15 units of T4 RNA ligase (GE Healthcare, Little Chalfont, Buckinghamshire, UK) at 16 °C for 2 h. Labelled samples were purified with MicroBio-Spin six columns (Bio-Rad, Richmond, CA, USA) and hybridized to microarrays at 55 °C with rotation at 20 rpm for 20 h. The arrays were scanned using an Agilent Microarray Scanner (G2565BA, Agilent Technologies). The scanned images were analysed using the Feature Extraction software, version 10.7.3.1 (Agilent Technologies) with background correction. Data analysis was performed with GeneSpring GX, version 12.6.0 (Silicon Genetics). Expression data were normalized to the 75th percentile using the GeneSpring normalisation option with no substantial difference in results. The miRNA microarray analysis was performed in duplicate for each cell line. Statistical comparisons were made with the use of both the GeneSpring analysis-of-variance tool and volcano plot filtering.

Katsushima K., Natsume A., Ohka F., Shinjo K., Hatanaka A., Ichimura N., Sato S., Takahashi S., Kimura H., Totoki Y., Shibata T., Naito M., Kim H.J., Miyata K., Kataoka K, & Kondo Y. (2016). Targeting the Notch-regulated non-coding RNA TUG1 for glioma treatment. Nature Communications, 7, 13616.

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Profiling Human miRNA Expression

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Pooled total RNA was dephosphorylated and labeled with pCp-Cy3 (Agilent Technologies) and T4 RNA ligase (GE Healthcare). Then, the labeled sample was purified using Micro Bio-Spin 6 columns (Bio-Rad) and hybridized to Human miRNA Microarray V3 kit (Agilent Technologies) platform containing 866 human and 89 human viral microRNAs documented in the Sanger miRbase (version 12.0). After 20 hours of hybridizations carried out at 55 °C, microarrays were washed and scanned using an Agilent scanner controlled by Agilent Scan Control software (version 7.0) and then analyzed with Agilent Feature Extraction software (version 9.5.3.1). The raw microRNA expression data were normalized using quantile normalization and analyzed with GeneSpring GX (Agilent Technologies, version 11.5) according to recommendations from Agilent Technologies.

Yuan Y., Liu X., Hao S., He Q, & Shen Z. (2020). Plasma levels of miR-143 and miR-145 are associated with coronary in-stent restenosis within 1 year of follow-up after drug-eluting stent implantation. Annals of Translational Medicine, 8(12), 756.

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Profiling Plasma Exosomal miRNAs in Mouse Model

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Total RNA containing miRNAs (100 ηg) from a total of 16 plasma exosome samples (SC(−): n = 8 and SF(−): n = 8) were reverse transcribed. Each sample was prepared according to the Agilent's miRNA recommended approach using the one-color technique. miRNA was dephosphorylated with calf intestine alkaline phosphatase (GE Healthcare Europe GmbH), denatured with dimethyl sulfoxide, and labeled with pCp-Cy3 using T4 RNA ligase (GE Healthcare Europe GmbH). The labeled miRNAs were hybridized to custom 8 × 60 K mouse microarrays, consisting of 60-mer DNA probes synthesized in situ that represent 2006 miRNA (Version 19.0) and each of them represented by 40 features probes (Agilent), Santa Clara, CA). After hybridization and washing, the arrays were scanned with an Agilent microarray scanner using high dynamic range settings as specified by the manufacturer. Microarray results were extracted using Agilent Feature Extraction software (v12.0). Bioconductor package AgiMicroRna [53 (link)] was used to perform the data preprocessing and limma [54 (link)] was also used for the differential expression analysis. The p-values were corrected for multiple testing by false discovery rate (FDR) using Benjamini-Hochberg procedure [55 ].

Khalyfa A., Almendros I., Gileles-Hillel A., Akbarpour M., Trzepizur W., Mokhlesi B., Huang L., Andrade J., Farré R, & Gozal D. (2016). Circulating exosomes potentiate tumor malignant properties in a mouse model of chronic sleep fragmentation. Oncotarget, 7(34), 54676-54690.

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Profiling miRNA Expression with Agilent Arrays

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We dephosphorylated and labeled pooled total RNA with pCp-Cy3 (Agilent Technologies) and T4 RNA ligase (GE Healthcare). Then the labeled sample was purified using Micro Bio-Spin 6 columns (Bio-Rad) and hybridized to Human miRNA Microarray V3 kit (Agilent Technologies) platform. After 20 h of hybridizations carried out at 55 °C, microarrays were washed and scanned using an Agilent scanner controlled by Agilent Scan Control software (version 7.0) and then analyzed with Agilent Feature Extraction software (version 9.5.3.1). The raw microRNA expression data were normalized using quantile normalization and analyzed with GeneSpring GX (Agilent Technologies, version 11.5) according to recommendations from Agilent Technologies.

Gao S., Xu Q., Zhou Y, & Yi Q. (2020). Serum levels of microRNA-21 and microRNA-10a can predict long-term prognosis in laryngeal cancer patients: a multicenter study. Translational Cancer Research, 9(5), 3680-3690.

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