Whole genome gene expression analysis was performed in paired sputum samples. RNA was extracted from induced sputum using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and quantitated using the Quant-iT RiboGreen RNA Quantitation Assay Kit (Molecular Probes Inc., Life Technologies, Carlsbad, CA, USA), where fluorescence was measured at wavelengths 485 nm (excitation) and 520 nm (emission) (FLUOstar Optima, BMG Labtech, VIC, Australia). A total of 500 ng of sputum RNA was amplified and labelled with biotin-uridine triphosphate using the Total Prep RNA amplification kit as per manufacturer’s instructions (Life Technologies). 750 ng cRNA was hybridised to Illumina Sentrix HumanRef-8 V3 Expression BeadChips (Illumina, San Diego, CA, USA) using standard protocols. Each BeadChip measured the expression of 24,354 genes and was scanned using the Illumina Bead Station and captured using BeadScan 3.5.11 (Illumina). The microarray primary data used in this study are available at the national center for biotechnology information (NCBI) Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/ ) under accession number GSE74075.
Quant it ribogreen rna quantitation assay kit
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
The Quant-iT RiboGreen RNA Quantitation Assay Kit is a fluorescence-based assay used to quantify RNA in solution. The kit provides a sensitive and accurate method for determining the concentration of RNA in samples.
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2 protocols using quant it ribogreen rna quantitation assay kit
1
Whole Genome Expression Analysis of Sputum
2
Adipose Tissue RNA Extraction Protocol
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The adipose tissue biopsies were thawed on ice and immediately homogenized in Qiazol reagent (Qiagen, Hilden, Germany) through high speed (25 Hz) shaking in the presence of a stainless steel bead for 2 min using the TissueLyser II (Qiagen). Chloroform was then added, and the homogenate was separated into aqueous and organic phases via centrifugation at 12,000 Â g for 15 min at 4 C. Total RNA was extracted from the aqueous phase using the RNeasy Mini Kit as per manufacturer's instructions (Qiagen). RNA concentration was determined using the Quant-iT RiboGreen RNA Quantitation Assay Kit (Molecular Probes Inc, Invitrogen, Eugene,USA), as per manufacturer's instructions. There was an average RNA yield of 23.8 ng/mL with a range of 0e117.2 ng/mL.
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