The cycling conditions were 3?min at 95?C, followed by 35?cycles of 30?s at 95?C, 30?s at 58?C, 1?min at 72?C, and finally 7?min at 72?C. carried out for each sample, analyzed using primer sequences reported in Table?1. The reactions were set on a strip in a final volume of 25?l by mixing, for each sample, 1?l of cDNA, 12.5?l of 2 concentrated SYBR Premix Ex Taq II (Takara Bio) containing SYBR Green as a fluorescent intercalating agent, 0.2?M forward primer, 0.2?M of reverse primer, and MQ water. PCR efficiencies were tested and found to be close to 1. The thermal profile for all reactions was 30?s at 95?C and then 40?cycles of 5?s at 95?C, and 30?s at 60?C. Fluorescence monitoring occurred at the end of each cycle. The efficiency of amplification for each primer was monitored through Ets1 the analysis of serial dilution. Additional dissociation curve analysis was performed, and in all cases showed a single peak. The data thus obtained were analyzed using the iQ5 optical system software version 2.0 (BioRad). The expression of each gene was normalized to the reference gene in order to standardize the results by eliminating variation in Complanatoside A cDNA quantity. Sequences used are listed in Table?1. miRNA analyses by RNA extraction and PCR amplification The MV pellet was subjected to RNase digestion to remove extraneous ribonucleic acids [41]. Total RNA was isolated from a pool of different MVs and amniotic-derived cell preparations using the NucleoSpin? mRNA kit (Macherey-Nagel, Germany), in combination with TRIzol? lysis and purification of small and large RNA in one fraction (total RNA). RNAs were quantified using a NanoDrop ND-1000 Complanatoside A spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA quality was checked using the Agilent Bioanalyser 2100 (Agilent, Santa Clara, CA, USA), where the presence of small RNAs was verified in both MV and cell samples. RNAs from all samples were reverse transcribed with the miScript Reverse Transcription Kit and the cDNA was then pre-amplified using the miScript PreAMP PCR Kit (all from Qiagen, Valencia, CA, USA), following the manufacturers instruction with some modification: miScript PreAMP Primer Mix was Complanatoside A replaced with miR-specific primers: hsa-miR-26a-2, -335, -146a, and SNORD95 as forward primer, and miScript Universal Primer as reverse primer in separate reactions. hsa miRNA were perfectly homologous with eca miRNA sequence. PCR was performed on pre-amplified products using the PCR Master Mix (2) (Thermo Fisher Scientific Inc., Waltham, MA, USA), with the same primer couple: hsa-miR-26a-2, -335, -146a, SNORD95 in combination with miScript Universal Primer. The small nucleolar snoRNA, C/D Box 95 SNORD95 was used as the positive control. Negative controls using water in place of the pre-Amp product were performed alongside each reaction. The cycling conditions were 3?min at 95?C, followed by 35?cycles of 30?s at 95?C, 30?s at 58?C, 1?min at 72?C, and finally 7?min at 72?C. The amplified PCR products were separated electrophoretically on 2.5?% agarose gels, and visualized under UV, using the GeneRuler 50?bp as a DNA ladder (Thermo Fisher Scientific Inc.). Cytokines Cytokine release (IL-6, transforming growth factor (TGF)-, and TNF-) was measured in cell-free supernatants obtained by Complanatoside A centrifugation at 1200?rpm for 5?min and stored at ?80?C until measurement. Cytokine production was assessed by commercially available sandwich ELISAs (Bioptis SA, Liege, Belgium). ELISAs were performed according to the suppliers instructions. Results are expressed in pg/ml. The limit of detection was 15.6?pg/ml for all cytokines tested. Statistical analysis For quantitative PCR experiments, data were analyzed by one-way analysis of variance (ANOVA). Also, cell viability data were analyzed by one-way ANOVA applying a Bonferroni correction. For cytokines, statistical differences were determined using ANOVA followed by Dunnetts multiple comparison test, the TukeyCKramer multiple comparisons test or unpaired test. Differences were considered statistically significant if the value of was 0.05. Results Tissue collection and cell isolation Cells were selected for their ability to adhere to plastic. For AMCs, the initial viability was 90?%, whereas for EDCs it was 85?%. EDCs (Fig.?1a) and AMCs (Fig.?1b) displayed fibroblast-like morphology. Molecular biology analyses at P3 showed that AMCs showed a typical mesenchymal stromal phenotype, with the expression of markers such as CD29, Complanatoside A CD44, CD106, CD105, and MHCI, but not CD34 and MHCII. Moreover, AMCs showed differentiative potential in mesenchymal (osteogenic, adipogenic, and chondrogenic) and ectodermic lines (neurogenic) as reported.