Selective real-time PCR assays were carried out using SABioscience kit (Qiagen, Inc.) using the amplified miRNA samples. As per the vendor’s protocol, we loaded 100 ng miRNA to 384-well plates containing anchored miRNA probes (50–75 bp; including miRNA sequence, tailing, and the universal primer) and the hybridization outcomes were quantified by the ABI HT 7900 real-time PCR system (Life technologies, Inc., Grand Island, NY). The vendor-recommended algorithm computed the relative miRNA expression level using the change of threshold cycle (Ct) i.e. 2 ^ (−Δ Ct), where Δ Ct = Ct (GoI) – avg. (Ct (HKG)). GoI represents the gene-of-interest, and HKG is the housekeeping gene. In order to eliminate the false positive candidates, the selected miRNA reads were screened by the following dual criteria applied together: (i) the control threshold cycle should be >30 and sample cycle <30 (or vice versa); and (ii) the p-value for the fold-change should be either unavailable or relatively high (p > 0.05) from the assay background.
From the pool of screened miRNA reads, we identified the probes that had different expressions (p < 0.05) between G-C vs. S-C. This cluster of miRNAs was altered exclusively by μG. Likewise, comparing G-4 h and S-4 h, the miRNA signatures of LPS assault mediated by μG were identified.
From the pool of screened miRNA reads, we identified the probes that had different expressions (p < 0.05) between G-C vs. S-C. This cluster of miRNAs was altered exclusively by μG. Likewise, comparing G-4 h and S-4 h, the miRNA signatures of LPS assault mediated by μG were identified.