Mirna v1 gene chip

Microarray for the miRNAs was performed at Johns Hopkins Memorial Institute (Deep Sequencing and Microarray Core Facility, Baltimore, MD, USA). Total RNA, including mRNA, miRNA and other small RNA molecules, were isolated from PBMC samples as described above. Next, total RNA samples were used in the analysis of miRNA expression level by miRNA array hybridization assay using the Affymetrix miRNA-v1 gene chip (Affymetrix, Sunnyvale, CA, USA). Linear fold-change in miRNA was calculated to compare the differences of all the miRNAs expressed between PTSD patients and controls. A linear fold-change of at least ±1.5 was used as a cutoff value for the inclusion of a miRNA. Moreover, only the miRNAs which were significant on the basis of P-value (<0.05) calculated using student’s t-test, were included for the analysis. For analyzing the miRNAs in the replication group, we combined all the data from both discovery and replication group. The miRNA array data are available in ArrayExpress (EMBL-EBI, Cambridgeshire, UK, Accession# E-MTAB-4880).

Total RNA, including miRNA and other small RNA molecules, were isolated from randomized PBMC samples of PTSD patients and normal controls, using miRNeasy Mini kit and following the protocol of the company (Qiagen, Valencia, CA). The RNA integrity was verified using Agilent 2100 BioAnalyzer (Agilent Tech, Palo Alto, CA). Next, total RNA samples were subjected to miRNA array hybridization assay using Affymetrix miRNA-v1 gene chip according to the manufacturer's instructions. The miRNA array data were analyzed with Affymetrix miRNA QC tool software. The analysis pipeline detected the probe signals, estimated background and correction, normalized the signals and summarized the signal using median polish. The array performance was assessed using the quality control probes on the array and Pearson correlation of the control probes across the arrays. Fold changes in miRNA up-regulation or down-regulation were calculated using the formula: [Fold change = IF(X2> = 0, 2∧X2, −1/(2∧X2)), X = A-B, A = raw signal of specific miRNA from a PTSD patient, and B = raw signal of specific miRNA from a control], to compare the differences in1163 miRNAs expressed between PTSD patients and normal controls.