The average age for HAD and non HAD patients was 51. 88 9. 45 and 43. 57 14. 77, respectively. Clinical profiles of all patients are shown in Additional 14. In order to make sure the quality of RNA, samples post mortem intervals were less than 48 hours have been selected for the study. Among them, Navitoclax chemical structure 5 samples from HAD and HIV non dementia group, respectively, have been randomly chosen for miRNA study. This study was conducted according to the principles expressed in the Declaration of Helsinki. Use of samples in this study was approved by the Institutional Review Board and the Eth ics Committee of the NNTC Allocations, the University of Sydney and the Westmead Hospital individually. The family members of the patients gave written, informed consent for the use of autopsied brain tissue.
For the diagnostic criteria for HAD, the criteria defined Inhibitors,Modulators,Libraries by the American Academy of Neurology 1991 were used. RNA isolation and mRNA and miRNA profiling Total RNA was extracted from 30 mg of brain cortex tissue. Tissue samples were homogenised using a high speed agitation polytron belnder in the presence of RNA lysis buffer. The RNA was iso lated and purified with a miRNeasy Mini kit with DNAse I digestion on the column according to the manufacturers protocol. The quality and quantitiy of the RNA preparations was assessed using an Agilent 2100 Bioanalyser. RNA in tegrity scores were 7 for all the samples analyzed. First Choice Human Brain Reference commercially available RNA was used as a control RNA for the microarray analysis.
For mRNA profiling, cRNA amplification and labeling with biotin were performed using Illumina TotalPrep RNA amplification Inhibitors,Modulators,Libraries kit according to the manufacturers directions with 500ng total RNA as input material. cRNA yields were quantified using Agilent 2100 Bioanalyser. Gene expression analysis was performed using the Sentrix Human 6 v2 Expression BeadChip, and BeadStation system from Illumina as per manufacturers Inhibitors,Modulators,Libraries instructions. The Human 6 v2 Expres sion BeadChip allows genome wide expression Inhibitors,Modulators,Libraries profiling of more than 48,000 gene transcripts and known alternative splice variants from the RefSeq database. For miRNA profiling, 1000ng total RNA was labelled with FlashTag Biotin HSR RNA Labeling Kit and analzed using Affymetrix GeneChip miRNA Array, which contains 1105 Homo sapiens miRNAs. Gene expression data analysis was performed using GenomeStudio version 3.
Inhibitors,Modulators,Libraries The gene expression data was normalised using the cubic spline function, the genes were selected if the detection P 0. 01 in selleck compound at least one group. All samples were coded and analyzed blindly to avoid any bias. The differential gene expression analysis was performed using Illumina custom error model with false discovery rate correction implemented in GenomeS tudio. Genes, whose DiffScore 13 or ?13, were considered statistically significant. miRNA data analysis was carried out using GeneSpring 11. 0.