Megaplex rt primers human pool

Total RNAs were extracted from P3-MSCs using the mirVana miRs isolation kit (Ambion, Thermo Fisher Scientific, USA) by following the manufacturer’s instructions. RNA concentrations and qualities were evaluated by using NanoDrop 2000 (Thermo scientific, USA). Total RNA samples, containing the fraction less than 200 nucleotides, were used for miRs profiling studies. Identical amounts of RNAs extracted from each patient and healthy control were pooled together and subjected (700 ng per RNAs’ pool) to qRT-PCR by using the TaqMan MicroRNA reverse transcription kit (Applied Biosystems, USA) and the Megaplex RT primers human pool (Applied Biosystems, USA). Subsequently, microfluidic cards TaqMan array human microRNA A + B v3.0 (Applied Biosystems, USA) were used, according to the manufacturer’s instructions. Three replicates for each pooled sample were analysed. MiRs’ expression levels were evaluated by comparative assay: samples were analysed on a ViiA7 (Applied Biosystems, USA) and data were processed by ViiA7 software and further elaborated by Expression Suite (v.1.0.3, Applied Biosystems, USA) also at the statistical level. 2^−ΔΔCt method was used to determine the relative miRs’ expression levels. U6 snRNA was used as endogenous control. Significant miRs expression changes were identified using a threshold of p < 0.05 (p value calculation based on 2^−dCt).

Total RNA was extracted using the miRVana miRNA isolation kit (Ambion, #AM1560, Austin, TX, USA). RNA quality and concentrations were analyzed on a NanoDrop M-1000 spectrophotometer and an Agilent 2100 Bioanalyzer. RNAs with quality scores >7.00 were used for expression assays. RNA concentrations were standardized to 200 ng/μL. TaqMan low-density miRNA arrays (TLDAs) (Applied Biosystems, #4444913, Foster City, CA, USA) were used to assess miRNA expression levels in proliferating basal cells grown in SAGM-EA. Reverse transcription of 600 ng total RNA was carried out using a TaqMan miRNA reverse transcriptase kit (Applied Biosystems, #4366596) with Megaplex RT primers, Human Pool (Applied Biosystems, #4399966). Samples were loaded onto the TLDA, which utilizes 384 wells preloaded with specific miRNA probes and primers in each well. The TLDA data were processed on an Applied Biosystems Model 7900 Genetic Analyzer, and the data were analyzed using the Applied Biosystems StatMiner software. Each sample was analyzed in triplicate, and each Ct value was normalized to the Ct value of RNU48 endogenous RNA control. Relative quantification of each miRNA was performed using the ΔΔCt method. Statistical significance of the fold change was assessed using two-tailed t-tests. p-values of <0.05 were taken as statistically significant.

5 patients were selected randomly for initial array experiments. RNA was converted to cDNA by priming with a mixture of looped primers (MegaPlex PreAmp Primers, Human Pool; MegaPlex RT primers, Human Pool; Applied Biosystems, Foster City, CA, USA). TaqMan Array miRNA Cards (#4444913, Applied Biosystems) were used containing a total of 384 unique assays specific to human miRNAs under standard qRT-PCR conditions. qRT-PCR was carried out on an Applied Biosystems 7900HT thermocycler using the manufacturer’s recommended program. Analysis of data was performed using Expression Suite V1.1 program (Applied Biosystems). Cycle of threshold (CT) values above 40 were defined as undetectable. U6snRNA was chosen as housekeeping gene, due to stable expression across all samples. CT values were exported to Microsoft excel (Microsoft Corporation, Albuquerque, NM, USA) and CT and ddCT levels were calculated. Evaluation of results was performed with GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, USA). Statistical analysis was performed using multiple T-Testing with Bonferroni-Dunn method for multiple comparisons and detection of false positives.