PBMC were isolated by gradient Histopaque® 1077 (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer's instructions. Total RNA was extracted from 5x106 cells with a miRVana™ miRNA Isolation Kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. RNA samples were immediately stored at -80ºC. Quantification was performed using NanoDrop (ND-2000 spectrophotometer, Thermo Scientific, Wilmington, DE, USA) and quality of samples was assessed using a Capillary electrophoresis (Bioanalyzer 2100 Agilent, Santa Clara, CA, USA).
Label
biotin
Label protocol
MicroRNA profiles were obtained from 250 ng/sample of total RNA (RNA Integrity Number over than 7) using the FlashTag™ Biotin HSR RNA Labeling Kit, and the Affymetrix® miRNA 4.1 Array strip (Affymetrix, Santa Clara, Califórnia, EUA), according to the manufacturer’s instructions. A recommended ELOSA quality control assay was run for all samples.
Hybridization protocol
For hybridization of samples to the strips was carried on at 48ºC for 20h using the GeneAtlas® Hybridization, Wash, and Stain Kit for miRNA Array Strips. Following this period, strips were processed and scanned using the GeneAtlas® System (Affymetrix).
Scan protocol
After the array strip are registered in the GeneAtlas Software and the target hybridization steps are complete, scanning processing on the GeneAtlas System was made. Transfer of the strip assembly into the imaging station's drawer starts the imaging process.
Description
miRNA profile expression in PBMC of dogs naturally infected with L. infantum
Data processing
Raw intensity values were background corrected, log2 transformed and then quantile normalized by the software Expression Console (Affymetrix) using the Robust Multi-array Average (RMA) algorithm.
