Taqman mir assay

qRT-PCR was performed to detect the expression levels of miR-328-3p in human tissues and cell lines according to our previous study.16 (link) Total RNA was extracted from fresh tissues and cells using TRIzol reagent (Thermo Fisher Scientific). TaqMan miR Assays (Thermo Fisher Scientific) with primers specific to miR-328-3p were used. Reverse transcription was performed using One Step PrimeScript miR cDNA Synthesis Kit (Thermo Fisher Scientific), and qRT-PCR was performed using SYBR Premix Ex Taq™ II (Thermo Fisher Scientific). RNU6B was used as an internal control. qRT-PCR primer sequences were as follows: miR-328-3p, forward 5′-CTG GCC CTC TCT GCC C-3′, and reverse 5′-GTG CAG GGT CCG AGG T-3′; RNU6B, forward 5′-ACA GUA GUC UGC ACA UUG GUU A-3′, and reverse 5′-ACG CAA ATT CGT GAA GCG TT-3′. Quantitative PCR was performed using an ABI 7500 Real-Time PCR Detection System (Thermo Fisher Scientific). The threshold cycle (Ct) was defined as the fractional cycle number at which the fluorescence passed the fixed threshold. Each sample was detected thrice, and the relative expression level of miR-328-3p to RNU6 was calculated using the equation 2^−ΔCt, where ΔCT = (CT_miR-328-3p/CT_RNU6). The median value of miR-328-3p expression in osteosarcoma tissues was used as a cutoff point for dividing miR-328-3p-low/high groups.

All samples were pre-amplified prior to use in the high-throughput quantitative real-time PCR instrument Biomark (Fluidigm Corporation). A preamplification pool was prepared from 20X TaqMan MiR Assays (Thermo Fisher Scientific, Inc.), each assay diluted at 1:100. A total of 10 µl of preamplification reaction contained: 2 µl of cDNA (not diluted), 1.5 µl of preamp pool, 5 µl of iQ Supermix (Bio-Rad Laboratories, Inc.) and 1.5 µl RNAse-free water. Preamplification was performed at C1000 (Bio-Rad Laboratories, Inc.) as follows: 95°C for 3 min, 18 cycles of 95°C for 15 sec and 59°C for 4 min. After preamplification, each reaction was diluted at 1:20.

MiRs were quantified with Taqman miR-Assays (all from Thermo Fisher Scientific: hsa-miR-30c Cat. # 4427975, Assay ID 000419; hsa-let-7d, Cat. # 4427975, Assay ID 002283; hsa-miR-20a, Cat. # 4427975, Assay ID 000580; hsa-miR-125a-3p, Cat. # 4427975, Assay ID 002199; RNU44, Cat. # 4427975, Assay ID 001094; snRNA U6 Cat. # 4440887, Assay ID 001973) in a 7500 HT Real-Time PCR instrument (Applied Biosystems). 10 ng of total RNA was reversely transcribed to cDNA by use of the TaqMan microRNA Reverse Transcription kit (Applied Biosystems, Cat. # 4366597), according to the manufacturer’s protocols. Then, quantitative real-time PCR was performed in triplicate using the TaqMan Universal Master Mix II (Applied Biosystems, Cat. # 4440040) and 1 µL of the cDNA solution after reverse transcription, which represents the equivalent of 0.67 ng RNA. Relative expression of miR-30c-5p, let-7d-5p and miR-20a-5p was calculated as ΔΔCT values, with RNU-44 as an internal control. Absolute miR expression was calculated by use of a concentration gradient of miR-30c-5p-mimic (Thermo Fisher Scientific, Cat. # 4464066 Assay ID MC11060) ranging from 6.67 × 10⁻¹⁰ to 6.67 × 10⁻¹⁷ g RNA and corresponding CT values from 6 to 39. MiR levels in EV-recipient cells were quantified after 24 h of incubation with EVs^{hnRNPU kd}/EVs^Control.