Taqman array human microrna card set v3

Total RNA (including small RNA) was extracted from 50 mg tissue samples with Direct-zol RNA Miniprep Plus kit (Zymo Research, Irvine, CA, USA); subsequently, RNA concentration was measured using a NanoDrop ultraviolet (UV) spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription was performed using TaqMan^® MicroRNA Reverse Transcription Kit with Megaplex™ RT Primers, Human Pool Set v3.0 (Thermo Fisher Scientific, USA) and 500 ng of total RNA in a reaction volume of 7.5 µL, separately for each of two pools of miRNA. Finally, quantitative PCR was performed in a ViiA7 Real-Time PCR System using TaqMan Array Human MicroRNA Card Set v3.0 (Thermo Fisher Scientific, USA). This set enables quantitation of 472 previously described human microRNAs and three housekeeping genes as endogenous controls (stably expressed small noncoding RNAs: U6 snRNA, RNU44, and RNU48) to aid in data normalization. The same threshold value was manually set for all targets, and only miRNA assays with Ct values ≤ 32 were further analyzed. The relative quantity (RQ) of each target was calculated using the ΔC_t method, in relation to mean expression of three endogenous controls.

miRNA expression analysis was performed by qRT-PCR array on three different samples of BMSC-EVs. Briefly, RNA was extracted from purified EVs by mirVana Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA), according with manufacturer’s instruction. RNA concentration was measured by Nanodrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA), and the ratio 260/280 and 260/230 showed absence of contaminants. Fifty nanograms of total RNA were retro-transcribed to cDNA with TaqMan^® MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was pre-amplified with Megaplex™ RT Primers, Human Pool Set v3.0 and TaqMan^® PreAmp Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) by using Veriti Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA). The expression profile of a panel of 754 human miRNAs was evaluated by TaqMan^® Array Human MicroRNA Card Set v3.0 (Thermo Fisher Scientific, Waltham, MA, USA) with QuantStudio12k Flex system (Thermo Fisher Scientific, Waltham, MA, USA). ExpressionSuite Software 1.1 (Thermo Fisher Scientific, Waltham, MA, USA) was used to calculate Ct values. Values of Amp score < 1.1 or Cq conf < 0.7 or Ct > 34 were excluded from analysis. Only miRNAs expressed in all samples of the same group were included for analysis.

We determined miRNA expression profile alteration in cell culture by TaqMan Array Human MicroRNA Card Set v3.0 (Thermofisher Scientific, Cat^#: 4444913) containing assays for a total 754 of human microRNAs as described before [28 (link)]. Individual miRNA expression was determined on the Viia 7 Real-Time PCR System (Life Technologies) [27 (link)] using the individual TaqMan assays for miR-1 (Assay ID: 000385).