Mircury lna microrna pcr system

miRNAs short listed as differentially expressed from the discovery set were revalidated in a larger cohort comprising of 145 individuals (50% men, 50% women) with NGT (n = 49), IGT (n = 47) and T2DM (n = 49) by individual PCR assays. Total RNA from serum was extracted as described above and used for qRT-PCR by using the Universal miRCURY LNA microRNA PCR, Polyadenylation and cDNA synthesis kit II and the miRCURY LNA microRNA PCR system, Exilent SYBR green master mix, from Exiqon, on ABI 7000 Applied Biosystems thermocycler. Relative expression of microRNA expression was calculated by 2^{ˆ(-"DeltaCt")} method. We evaluated a suitable number of reference miRNAs, based on the increased expression stability [28 (link)]. However, we were unable to identify stable miRNAs that did not discriminate between male and female or that were not correlated with metabolic parameters, or that was not previously identified as relevant for the pathology in previous studies. This could be explained partly by the fact that inside the same group, women and men might differed in their level of cholesterol and triglycerides (S1 Table). Thus, in this study, data were normalised on the volume of total RNA that was used for qRT-PCR (i.e.; a fixed volume of 8 μl).

A 1–4 ng aliquot of total RNA was then subjected to cDNA synthesis using either miRNA- (miR-150- and miR-146a-) or U6 snRNA-specific primers. 5S rRNA amplifications using hexamer-primed cDNA as template were performed as positive controls. All reactions were performed using the miRCURY LNA™ microRNA PCR system (Exiqon, Denmark) according to the manufacturer's instructions [22 (link)]. The cycling conditions were 95°C for 10 min, followed by 42 cycles at 95°C for 20 s and 60°C for 1 min. All cycles were completed on an Mx3005P cycler (Stratagene, USA). The miRNA expression levels were measured using SYBR green qRT-PCR, normalised to those of U6 snRNA, and calculated by the comparative CT method.

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).

Harvested tissues were homogenized by TissueLyzer II (Qiagen, Hilden, Germany), and total miRNAs and mRNAs were extracted/purified using an miRNeasy Mini Kit (Qiagen) and RNeasy Plus Universal Mini Kit (Qiagen), respectively, according to the manufacturers' instructions. Quantification of miRNAs was performed by TaqMan MicroRNA Assay (Applied Biosystems, Foster City, CA, USA) and the miRCURY LNA microRNA PCR system (Exiqon, Vedbaek, Denmark) using ABI PRISM 7900HT (Applied Biosystems). Quantification of mRNAs was performed with Thunderbird SYBR qPCR Mix (Toyobo Inc., Osaka, Japan) using ABI PRISM 7900HT (Applied Biosystems) (sequences of primers are shown in Appendix Table S6).

Verification of the miRNA microarray analysis was conducted using the miRCURY LNA™ microRNA PCR system (Exiqon) according to manufacturer's instructions. For each miRNA assay, three separate cDNA syntheses were performed for each of two biological replicates followed by a qPCR reaction. Quantitative Reverse Transcription PCR (qRT-PCR) was performed using an Applied Biosystems 7500 PCR Instrument (Applied Biosystems, Foster City, CA, USA). To verify specificity and identity of the miRNAs, a melting/dissociation curve was conducted for each miRNA after the qRT-PCR run. The baselines of the qRT-PCR runs were automatically set by the PCR instrument, while the threshold for fluorescence was manually set in the exponential phase. The inclusion criteria of Ct values were as follows: Ct value between replicates below 0.5, and amplification of at least two replicates. Furthermore, the quality of the qRT-PCR run was analyzed by examination of the amplification plot and the melting temperature curve. The stability of 5 candidate reference genes was tested using qBase v.1.3.4 [48 (link)] and geNorm [49 ] to identify the most stable reference genes in the three cell lines. The qRT-PCR data was normalized and analyzed using the ΔΔCt method [50 (link)]. ΔCt values were normalized using the geometric mean of the three most stable endogenous reference genes, U6 snRNA, 5S rRNA and hsa-miR-191.

qPCR validation was carried out on a LightCycler 480 Instrument (Roche) using the miRCURY LNA microRNA PCR system (Exiqon) for miRNA expression. All qPCR reactions were performed in duplicate. U6 snRNA and 5s rRNA were used as internal reference controls. qPCR validation of mRNA expression was performed using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche Diagnostics) and SYBR qPCR Master Mix (Agilent Technologies) and gene-specific primers (Supplementary Table 1). All qPCR reactions were performed in duplicate and normalized to internal housekeeping genes, Atp5b and Rpl10.