Miscript mirna pcr system

For validation of miRNA expression in infected macrophages (saprophyte, attenuated and virulent strains) and non-infected control macrophages, we employed the miScript miRNA PCR System (Qiagen-Valencia, CA, USA) for preparation of cDNA and realtime PCR, according to manufacturer´s instructions. Validated inventoried primers employed were purchased from Qiagen. PCR was performed using a Stratagene QPCR System Mx3005P (Agilent Technologies, Santa Clara, CA, USA), following instructions on the miScript miRNA PCR System´s manual. Expression levels were determined using standard curves for all miRNAs at each individual run, and the expression of candidate miRNAs is presented as a ratio to the control miRNA SNORD96A.

Relative expression levels of mature miRNA were determined using the miScript miRNA PCR System (Qiagen). Total RNA was subjected to reverse transcription using the miScript II RT Kit (Qiagen) while the miScript SYBR^® Green PCR Kit (Qiagen) and miScript primer assays were used for PCR amplification, according to manufacturer’s recommendations. The following miScript primer assays were used: MS00031549 (for hsa-miR-193b-3p), MS00008939 (for hsa-miR-193b-5p), MS00031801 (hsa-miR-365a-3p), and MS00041762 (hsa-miR-365a-5p), as well as MS00033740 (for snRNA RNU6B) that served as reference. The specificity of qRT-PCR amplification was confirmed with dissociation curve analysis.

High-throughput RT-qPCR was carried out on three different systems: TaqMan miRNA PCR system (Applied Biosystems, California, USA), miRCURY LNA microRNA PCR system (Exiqon) and miScript miRNA PCR system (Qiagen). The RT, preamplification and qPCR reactions were carried out according to previous publication31 (link) and as indicated in Supplementary Table S8 and Supplementary Table S9. Nuclease free water was used as negative control. Samples from serial dilution of pooled synthetic miRNAs (Integrated DNA Technologies) and pooled cell line RNAs were used to construct standard curves and titration curves, respectively. All negative controls, serial diluted samples for standard curves and titration curves were included in all RT, preamplification and qPCR steps. All miRNA assays were performed in triplicate wells. High-throughput miRNA profiling was performed using the 48.48 or 96.96 Dynamic Array™ integrated fluidic circuits and run on the BioMark™ System (Fluidigm, California, USA). Correlation of preamplified samples with non-preamplified samples were also evaluated using six miRNA assays in the 96-well plate format and run on the ABI7500 FAST System (Applied Biosystems).