Serum miRNA profiling by quantitative RT PCR [Validation]

Initial screening, the expression of 125 mature miRNA was compared between pooled control and autism samples by two microarray analysis. The differential expression of 14 miRNA was further validated by SYBR green quantitative PCR of Individual samples. Thirteen miRNAs were differentially expressed in autistic individuals compared to the controls (8 upregulated and 5 down regulated). miR-151a-3p, miR-181b-5p, miR-320a, miR-328, miR-433, miR-489, miR-572, and miR-663a were downregulated, while miR-101-3p, miR-106b-5p, miR-130a-3p, miR-195-5p, and miR-19b-3p were upregulated. We have identified a set of serum miRNAs that could be used as non-invasive biomarkers for autism. SYBR Green qPCR, performed on an ABI PRISM 7900 SDS (ABI), was used for the validation of differentially expressed 14 miRNAs. Ten microliters of qPCR reaction mixture was prepared with a universal primer, primer assay, Template cDNA and RNase free water. All the qPCR reactions were performed in triplicate with the following cycling conditions: 95ºC →15 min, followed by 40 cycles of 94ºC →15 sec, 55ºC →30 sec and 70ºC → 30 sec.