microRNA profiling in different stages of rheumatoid arthritis

Determine the comprehensive miRNA expression profile in RNA extracted from whole blood samples from healthy controls (CTRL), individuals positive to CCP antibodies >25UI/ml (CCP+), early rheumatoid arthritis (ERA) and established rheumatoid arthritis (CRA) according to the ACR/EULAR 2010 classification criteria. Several miRNAs were overexpressed on ERA patients. We selected 5 miRNAs (miR-361-5p, miR-23a-3p, miR-223-3p, miR-4634 and miR-128-3p) for its quantification on a bigger group of subjects by RT-qPCR and the results showed good concordance with microarray expression data. A convenience sampling for the study was made with Individuals recruited in rheumatology clinics between January 2013 and November 2017. We collected 4 to 5 ml of whole blood in EDTA tubes plus 1ml of RNA later and homogenized immediately to prevent changes in their transcriptional profiles. Samples were stored at -70°C until processed for RNA extraction. The groups were divided according to classification criteria by ACR/EULAR 2010 by certified clinical rheumatologists. For the stratification of early and established RA the main criteria was the symptoms persistence for less or more than one year respectively. Healthy controls were considered as individuals with no relevant medical history, normal results on the COPCORD questionnaire, CCP levels <25U/ml and a normal ESR. The criteria for CCP+ group was CCP values >25U/ml and no RA clinical symptoms. A sample of whole blood for each subject was mixed with Tri-Pure reagent (ROCHE, USA) and centrifuged at 13,000xg for 15 minutes at 4°C. The aqueous phase was then transferred to a pre-filter column of Absolutely-miRNA RNA kit (Agilent) following the manufacturer’s instructions. 100ng of total rna were used for microarray analysis.