Flashtag biotin hsr rna labelling kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The FlashTag Biotin HSR RNA Labeling Kit is a tool used in the process of labeling RNA molecules with biotin for various research applications. The kit provides the necessary reagents and protocols to efficiently label RNA samples with biotin, enabling their subsequent detection and analysis.

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FlashTag™Biotin HSR kit (3 citations) FlashTag Biotin HSR Labelling Kit (2 citations) FlashTag BioTin RNA labelling kit (2 citations)

16 protocols using flashtag biotin hsr rna labelling kit

Affymetrix miRNA Expression Profiling

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600–900 ng of total RNA was labelled using the Affymetrix FlashTag Biotin HSR RNA labelling kit according to the manufacturer's instructions. Following FlashTag labelling, the biotin-labelled samples were stored at −20°C prior to hybridisation onto Affymetrix GeneChip miRNA 4.0 for 17.5 hours at 48°C 60 rpm in an Affymetrix hybridisation oven 645.
Following hybridisation, the arrays were washed using Affymetrix hybridisation wash and stain kit on the GeneChip Fluidics station 450 using fluidics script FS450_0002 and scanned using the Affymetrix GeneChip scanner 3000 7G.

Balaskas P., Goljanek-Whysall K., Clegg P., Fang Y., Cremers A., Emans P., Welting T, & Peffers M. (2017). MicroRNA Profiling in Cartilage Ageing. International Journal of Genomics, 2017, 2713725.

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Microarray Analysis of hiPSC miRNA

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Undifferentiated hiPSCs were maintained on Matrigel (Corning, New York, NY, USA) -coated dishes in mTeSR1 medium (StemCell Technologies, Vancouver, Canada). Total RNA was isolated from hiPSC lines using a miRNeasy mini kit (Qiagen, Hilden, Germany) and treated with DNase I according to the manufacturer’s instructions. Total RNA containing low molecular weight RNA (from six hiPSC lines of the training set, n = 6 for each line) were labelled using the FlashTag Biotin HSR RNA labelling kit (Affymetrix, Sunnyvale, CA, USA), according to the manufacturer’s instructions. Labelled RNA was processed for microarray hybridisation to miRNA 3.0 array (Affymetrix). An Affymetrix GeneChip fluidics station was used to perform streptavidin/phycoerythrin staining. The hybridisation signals on the microarray were scanned using a GeneChip Scanner 3000 (Affymetrix), and normalisation was performed using the miRNA array RMA + DABG analysis and the Expression Console software (Affymetrix). The National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) accession number for the miRNA array data is GSE117739.

Ohashi F., Miyagawa S., Yasuda S., Miura T., Kuroda T., Itoh M., Kawaji H., Ito E., Yoshida S., Saito A., Sameshima T., Kawai J., Sawa Y, & Sato Y. (2019). CXCL4/PF4 is a predictive biomarker of cardiac differentiation potential of human induced pluripotent stem cells. Scientific Reports, 9, 4638.

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HUVEC-MDA-MB-231 Nanobridge Transfer Protocol

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DiL-Ac-LDL-labelled HUVEC and CFSE-labelled MDA-MB-231 cells were co-cultured in Matrigel using standard co-culture protocol for 36 h. The cells were harvested and stained with PECAM-1 (1:25) (Abcam) and Alexa Fluor 647 (1:100) (Life Technologies) secondary antibodies. Stained cells were then FACS sorted into PECAM-1 + /DiL-Ac-LDL + /CFSE + and PECAM-1 + /DiL-Ac-LDL + /CFSE − populations, capturing HUVECs that did and did not receive nanobridge-mediated transfer. The sorted cells were then pelleted and snap frozen in liquid nitrogen. Total RNA isolation was carried out using the Ambion mirVana RNA Isolation Kit (Life Technologies) as per the manufacturer’s protocol and quantified using NanoDrop (Thermo Scientific). An miRNA microarray was carried out on two sets of the above mentioned samples from independent experiments with HUVECs stained with 7 μM CFSE as control. The Affymetrix GeneChip miRNA 3.0 Array was used. Labelling was carried out using the Affymetrix Flashtag Biotin HSR RNA labelling kit and standard protocol with a 1:500 dilution of ATP for Poly(A) Tailing. Hybridization was carried out using the Affymetrix GeneChip Hybridization Oven 640 for 42 h. Microarray data analysis was carried out using the bioinformatics toolbox in MATLAB (MathWorks); GEO accession number GSE72679.

Connor Y., Tekleab S., Nandakumar S., Walls C., Tekleab Y., Husain A., Gadish O., Sabbisetti V., Kaushik S., Sehrawat S., Kulkarni A., Dvorak H., Zetter B., R. Edelman E, & Sengupta S. (2015). Physical nanoscale conduit-mediated communication between tumour cells and the endothelium modulates endothelial phenotype. Nature Communications, 6, 8671.

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Robust Microarray-based miRNA Profiling

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Total RNA was extracted from triplicate biological replicate samples using the Qiagen miRNeasy kit according to the manufacturer’s instructions. RNA quantity was assessed using the NanoDrop ND-1000 spectrophotometer and RNA quality assayed using the Agilent RNA 6000 NANO KIT and Agilent Bioanalyzer (Agilent, Santa Clara, CA, United States). The FlashTag Biotin HSR RNA Labelling Kit (Affymetrix, Santa Clara, CA, United States) was used to label a total of 500 ng of total RNA according to manufacturer’s procedure. The GeneChip Scanner 3000 7G System and reagents from Affymetrix were used to hybridize, wash, stain and scan the Genechip miRNA 3.0 arrays (Affymetrix, Santa Clara, CA, United States).

O’Sullivan F., Keenan J., Aherne S., O’Neill F., Clarke C., Henry M., Meleady P., Breen L., Barron N., Clynes M., Horgan K., Doolan P, & Murphy R. (2017). Parallel mRNA, proteomics and miRNA expression analysis in cell line models of the intestine. World Journal of Gastroenterology, 23(41), 7369-7386.

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Gene and miRNA Expression Profiling

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RNA extracts were prepared using the Affymetrix WT PLUS Reagent Kit (Affymetrix). RNA quality and quantity were assessed using a 2100 Agilent Bioanalyzer (Agilent) and a NanoDrop instrument (Thermo Scientific), respectively. For gene expression analysis, 100 ng of total RNA was used in conjunction with the Affymetrix standard protocol for Human Transcriptome Arrays 2.0 (Affymetrix Inc.). For the miRNAs analysis, 1 μg of total RNA was analysed using the FlashTag Biotin HSR RNA labelling kit for the Affymetrix Genechips miRNA 4.0 microarrays (Affymetrix Inc.).

Shah P., Fritz J.V., Glaab E., Desai M.S., Greenhalgh K., Frachet A., Niegowska M., Estes M., Jäger C., Seguin-Devaux C., Zenhausern F, & Wilmes P. (2016). A microfluidics-based in vitro model of the gastrointestinal human–microbe interface. Nature Communications, 7, 11535.

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Microarray Analysis of miRNA Expression

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The FlashTag Biotin HSR RNA labelling Kit (Affymetrix, High Wycombe, United Kingdom) was used to label 500 ng of total RNA. The biotin labelled samples were hybridized to GeneChip miRNA 3.0 arrays (Affymetrix, High Wycombe, United Kingdom) using a hybridization cocktail. Microarray hybridization was performed in an Affymetrix GeneChip Hybridization Oven-645 for 16 h at 48 °C and 60 rpm. Hybridized arrays were washed and stained using the GeneChip HWS Kit (Affymetrix, High Wycombe, United Kingdom) and the fluidics station protocol (FS450_0002) on an Affymetrix GeneChip Fluidics Station-450. The stained arrays were scanned with an Affymetrix GeneChip Scanner-3000-7G, and quality of the scanned arrays was evaluated using Affymetrix GCOS software. The generated raw data files were used for data analysis.

Chaudhari U., Nemade H., Gaspar J.A., Hescheler J., Hengstler J.G, & Sachinidis A. (2016). MicroRNAs as early toxicity signatures of doxorubicin in human-induced pluripotent stem cell-derived cardiomyocytes. Archives of Toxicology, 90(12), 3087-3098.

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Microarray Analysis of Cartilage miRNA Profiles

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RNA was extracted from cartilage^{23 (link)}. Twenty-nine Affymetrix miRNA-4.0 microarrays were used. Total RNA samples were quantitated using a Nanodrop spectrophotometer (NanoDrop Technologies). 500 ng of total RNA was labelled using the Affymetrix Flash-Tag Biotin HSR RNA labelling kit according to manufacturer’s instructions. Following Flash-Tag labelling the biotin-labelled samples were stored at − 20 °C prior to hybridisation onto Affymetrix GeneChip miRNA 4.0 for 17.5 h at 48 °C 60 rpm in an Affymetrix hybridisation oven 645. Following hybridisation the arrays were washed using Affymetrix Hybridisation wash and stain kit on the GeneChip Fluidics station 450 using fluidics script FS450_0002, and scanned using the Affymetrix GeneChip scanner 3,000 7G. CEL files were generated using the Affymetrix GeneChip Command Console Software, and Expression Console software was used to QC.

Peffers M.J., Chabronova A., Balaskas P., Fang Y., Dyer P., Cremers A., Emans P.J., Feczko P.Z., Caron M.M, & Welting T.J. (2020). SnoRNA signatures in cartilage ageing and osteoarthritis. Scientific Reports, 10, 10641.

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Biotin-Labelled miRNA Profiling

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Total RNA was isolated from frozen tissue using miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction before evaluated by the NanoDrop2000 spectrophotometer (Thermo Scientific, MA, USA). Total RNA was purified and labelled using FlashTag™ Biotin HSR RNA Labelling Kit (P/N 901,911, Affymetrix) according to the manufacturer’s instructions to obtain biotin labelled miRNA. Array hybridization and wash were performed by GeneChip® Hybridization, Wash and Stain Kit (P/N900720, Affymetrix Santa Clara CA, USA) and GeneChip Eukaryotic Hybridization Control Kit (P/N 900,454, Affymetrix Santa Clara CA, USA) in Hybridization Oven 645 (P/N 00–0331 (220 V), Affymetrix Santa Clara CA, USA) and Fluidics Station 450 (P/N 00–0079, Affymetrix Santa Clara CA, USA) according to the manufacturer’s instructions. Arrays were scanned by GeneChip® Scanner 7G (Affymetrix, Santa Clara, CA, USA) using Command Console Software 3.2 (Affymetrix, Santa Clara, CA, USA) with default settings. Raw data was normalized by RMA and DABG algorithm, Expression Console (Affymetrix, Santa Clara, CA, USA).

He S., Huang Y., Dong S., Qiao C., Yang G., Zhang S., Wang C., Xu Y., Zheng F, & Yan M. (2020). MiR-199a-3p/5p participated in TGF-β and EGF induced EMT by targeting DUSP5/MAP3K11 in pterygium. Journal of Translational Medicine, 18, 332.

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miRNA Expression Profiling using GeneAtlas Platform

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Total RNA (130 ng) enriched for low molecular weight RNA from each sample was labelled using the FlashTag Biotin HSR RNA Labelling Kit (Affymetrix) on the GeneAtlas Hybridization Station (Affymetrix) and subsequently, it was processed using the GeneAtlas Hybridization, Wash and Stain Kit for miRNA Array Strips (Applied Biosystems, USA) on the GeneAtlas Fluidics Station (Affymetrix) according to the manufacturer’s instructions. Array strip fluorescence intensities were finally determined using the GeneAtlas Imaging Station (Affymetrix). Raw data were processed and visualized using Partek Genomics Suite software (Partek, USA).

Zedníková I., Chylíková B., Šeda O., Korabečná M., Pazourková E., Břešťák M., Krkavcová M., Calda P, & Hořínek A. (2020). Genome-wide miRNA profiling in plasma of pregnant women with down syndrome fetuses. Molecular Biology Reports, 47(6), 4531-4540.

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Microarray Analysis of miRNA Expression

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MiRNA expression patterns were examined by GeneChip miRNA 4.0 arrays (Affymetrix, USA), which was designed based on miRBase version 20. The array included 1908 probes for mature microRNAs and 1255 for pre-mature microRNAs. One microgram of total RNA was used as input into the labelling reaction as recommended by the protocol of the FlashTag Biotin HSR RNA Labelling Kit (Affymetrix, USA). Labelled miRNA was then hybridized to the array for 16 hours at 48 °C and 60 rpm. Then Genechips were scanned by using the GeneChip Scanner 3000 7 G (Affymetrix, USA).

Copier C.U., León L., Fernández M., Contador D, & Calligaris S.D. (2017). Circulating miR-19b and miR-181b are potential biomarkers for diabetic cardiomyopathy. Scientific Reports, 7, 13514.

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