Taqman microrna reverse transcription kit reagent

For miRNA expression analysis, 10 ng of RNA was mixed with TaqMan MicroRNA Reverse Transcription Kit reagent containing specific miRNA primers and reverse-transcribed according to the manufacturer’s instructions (Thermo Fisher Scientific, Vilnius, Lithuania). Real-Time PCR was performed by diluting the complementary cDNA product in 2x TaqMan Universal Master Mix II (with no AmpErase UNG) and 20x TaqMan microRNA Expression Assay for each mature miRNA to be measured: miR-21-5p (assay ID: 000397), miR-320e (assay ID: 243005_mat), and miR-4454 (assay ID: 461830_mat). Real-time PCR was performed in triplicate for each sample and each miRNA using the ABI PRISM 7900HT platform. Fold changes in miRNA relative to the U6 (assay ID: 001973) and cel-miR-39 (assay ID: 000200) endogenous controls were calculated using the 2^−ΔΔCt method.

Total RNA was isolated from cells and exosomes using the miRNeasy Micro kit according to the manufacturer's instructions (Qiagen Inc., Valencia, CA). RNA concentration was measured using the NanoDrop ND-100 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). For miRNA expression analysis, 50 ng of RNA was mixed with TaqMan MicroRNA Reverse Transcription Kit reagent containing specific miRNA primers and reverse transcribed according to the manufacturer's instructions (Thermo Scientific, Rockford, IL). Real-time PCR was performed by diluting the complementary cDNA product in 2× TaqMan Universal Master Mix II and 20× TaqMan microRNA Expression Assay for each mature miRNA to be measured: miR-1305 and miR-21 (Applied Biosystems, Waltham, MA). U6 and cel-miR-39 were used as the normalization controls for cells and extracellular vesicles, respectively, in miRNA expression-level analysis. For quantification of gene expression, 2 μg RNA was converted to cDNA using an Omniscript RT kit (Qiagen, Valencia, CA). The cDNA generated was amplified using TaqMan assay for HIF-1α, Nanog, OCT4, SOX2 and β-actin. Reactions were carried out in a QuantStudio 6 system (Applied Biosystems) and the results expressed as fold change calculated with the comparative CT (ΔΔCt) method relative to the control sample. β-Actin was used as the internal control for mRNA analysis.