Cultured cells were washed tree times with sterile phosphate buffer solution (pH 7,2) for removal of antibiotics and non-adherent cells. L. interrogans and L. biflexa were harvested by centrifugation and the pellet was resuspended in RPMI-1640 media (Sigma®, USA), and 100:1 bacteria:cell were added to macrophages at >90% confluency. Treatments, performed in triplicates, were carried as follows: infection of macrophages with a virulent strain (L. interrogans), infection with attenuated strain (L. interrogans), saprophyte strain (L. biflexa) and non-infected macrophages (controls). All treatments were incubated in fresh RPMI medium, without antibiotics, for 8h at 37º C, 5% C02. Following this period, RNA extraction was immediately performed as described below.
Growth protocol
Murine macrophage cell line J774A.1, provided by the Paul Ehrlich cell bank, Rio de Janeiro, Brazil, was maintained in RPMI-1640 media (Sigma®, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, USA), 100ug/mL streptomycin (Sigma Chemical Co St.Louis, MO), 0.03% L-glutamine solution (Sigma) and 100 UI/mL of penicillin. Cells were incubated at 37º C, 5% CO2 until formation of a confluent monolayer in 6-well plates (3cm wells).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from macrophages 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 capillary electrophoresis (Bioanalyzer 2100 Agilent, Santa Clara, CA, USA).
Label
biotin
Label protocol
MicroRNA profiles were obtained from 250 ng/sample of total RNA (RIN 10) 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
Hybridization of samples to the strips was carried out at 48ºC for 20h in a GeneAtlas™ Hybridization Oven, according to the instructions of the GeneAtlas® Hybridization, Wash, and Stain Kit for miRNA Array Strips.
Scan protocol
Following hybridization, wash and stain of strips were performed using the GeneAtlas® Hybridization, Wash, and Stain Kit for miRNA Array Strips in the GeneAtlas Robotic Wash Station. Following, strips were and scanned using the GeneAtlas® Imaging System (Affymetrix).
Description
miRNA profile expression of macrophages non-infected, at 8h
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.
