Flashtag biotin hsr labeling kit

Endobronchial biopsies from the GLUCOLD study were immediately snap-frozen and stored at −80 °C. RNA was extracted using the miRNeasy mini kit (Qiagen) according to the manufacturer’s protocol. Purity of RNA fractions was checked on NanoDrop 1000 UV-Vis spectrophotometer and RNA integrity was verified using RNA 6000 Pico Assay RNA chips run on an Agilent 2100 Bio analyzer (Agilent Technologies, Palo Alto, CA). Microarray hybridization was performed at the Boston University Microarray Resource Facility as described in FlashTag™ Biotin HSR Labeling Kit (Affymetrix, Santa Clara, CA, current version available at www.affymetrix.com).

Total RNA was collected from the exosomes purified as described above using TRI Reagent (Molecular Research Center, Inc). After DNase treatment, the RNA was labeled using a FlashTag Biotin HSR labeling kit (Affymetrix) according to the manufacturer’s instructions. The resultant mix of each sample was applied to an array on the Affymetrix GeneChip miRNA 4.0 Arrays (Affymetrix). Next, the probe arrays were washed, stained, scanned, and analyzed using the Affymetrix GeneChip Fluidics Station 450.

Twenty-four RNA samples (150 ng in 8 μL) isolated from young BC tissue samples were labeled using the FlashTag™ Biotin HSR Labeling Kit and later hybridized to the GeneChip miRNA 3.0 Array (Affymetrix Inc., Santa Clara, CA, USA) in accordance with the manufacturer’s instructions. Data were analyzed within the R statistical environment using Bioconductor (http://www.bioconductor.org (accessed on 6 January 2022)) packages, as mentioned previously [12 (link)].

For analysis with Affymetrix GeneChip miRNA Arrays (Affymetrix, Santa Clara, CA, USA), each sample was prepared with 150 ng of total RNAs. Samples were labeled with the FlashTag Biotin HSR labeling kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer's instructions. The RNA sample was mixed into the Poly A Tailing master mix, and FlashTag™ Biotin HSR Ligation was performed by adding FlashTag Biotin HSR Ligation Mix to each of the tailed RNA samples. T4 DNA Ligase was added to each sample for labeling reaction. The hybridization cocktail was then added to each labeled sample, the resultant mix of each sample was applied to an array on the Affymetrix GeneChip miRNA 4.0 Arrays, the probe arrays were washed and stained, then scanned and analyzed using the Affymetrix GeneChip Fluidics Station 450. Differentially expressed miRNAs were identified using fold changes as well as P values calculated by the Student's t-test. The thresholds set for up and down regulated miRNAs were a fold change ≥2.0 and P≤0.05.