Taqman microrna assay primers

For RNA isolation from cells or EVs (experiments in Section 2.1, Section 2.2, Section 2.3 and Section 2.4), total RNA was extracted with the miRNeasy mini kit (Qiagen) following the manufacturer’s protocol. For EV samples, 6.25 fmol of Cel-miR-39 and cel-miR-238 were spiked into 5 µg of purified EVs before performing the RNA extraction. TaqMan assays were used to assess miRNA expression; 10 ng RNA were reverse-transcribed into cDNA using the TaqMan microRNA Reverse Transcription kit and the TaqMan microRNA assay primers (Applied Biosystems, Foster City, CA).). qPCR was performed with the resulting cDNA using the TaqMan microRNA primers and TaqMan Universal PCR master mix reagents (Applied Biosystems). Thermal cycling was performed on an Applied Biosystems 7900 HT detection system (Applied Biosystems). For cells, the relative miRNA levels were normalized to RNU44 and RNU48, two internal controls. For EVs, the relative miRNA levels were normalized to cel-miR-39 and cel-miR-238, two spiked-in miRNAs.

cDNA, previously pre-amplified, was diluted 1:10 in Tris-EDTA (TE) buffer 1× and added to a final qRT-PCR reaction volume of 20 µL, which contained TaqMan MicroRNA assay primers (Applied Biosystems) for each miRNA (Table S1), TaqMan Gene Expression Master Mix (2×) (Applied Biosystems), and nuclease-free water. The reaction was performed using ABI 7500 fast real-time PCR systems (Applied Biosystems) at 95 °C for 10 min and 40 cycles at 95 °C for 15 s and 60 °C for 60 s.
After validating the Ct mean of the housekeeping genes, U6snRNA, miR-16, and miR-1228, as reference miRNAs, the relative levels of each specific miRNA was calculated using the equation 2^-∆Ct, where ∆Ct = mean Ct_miRNA-mean Ct_{(miR-U6,16&1228)}, and Ct = threshold cycle.

Total RNA was isolated from clinical tissue samples with a total RNA isolation kit (AP-MN-MS-RNA, Axygen, CA, USA) as described by the manufacturer. Total RNA from each cell was isolated with Trizol Reagent (Invitrogen, NY, USA) and miRNAs were isolated with microRNA Purification Kit (Norgen Biotek, Thorold Ontario, Canada), according to the manufacturer’s protocol. miRNA-specific quantitative PCR was performed with Taqman MicroRNA Assay primers (Applied Biosystems) according to the manufacturer’s instructions. The miRNA level was normalized by U6 RNA. The qPCR for mRNA was performed with SYBR Green PCR Master Mix (Applied Biosystems).

Reverse transcription of mRNA used 250 ng of total RNA and was performed according to manufacturer’s instructions using GoScript reverse transcription kit with random hexamer primers (Promega). Quantitative PCR was performed in triplicate on a Rotor Gene 6000 (Qiagen) using GoTaq SYBR Green Mastermix (Promega) according to manufacturer’s instructions. Primer sequences used to confirm gene expression are provided in Additional file 8: Table S5. Data were normalized to the housekeeping gene ppia and relative expression was calculated using the 2^-ΔΔCt method [93 (link)].
Reverse transcription of miRNA used 10 ng of total RNA and was performed according to manufacturer’s instructions using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) and TaqMan MicroRNA Assay primers for mature miR-29b, miR-223 and U6 snRNA (Assay ID 000413, 000526, 001973, respectively; Applied Biosystems). Quantitative PCR was performed using TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems) according to the manufacturer’s instructions. Levels of miR-29 and miR-223 were normalized to U6 snRNA and relative fold change between control and crush tissue was determined using 2^-ΔΔCt method.

The expression of miR-125b and RNU48 (endogenous control) was measured by qPCR. Total RNA was converted to cDNA using the TaqMan microRNA reverse transcription kit and the TaqMan MicroRNA assay primers (all from Thermo Fisher, Monza, Italy) according to the manufacturer's instructions. The mixture was incubated at 16 °C for 30 min, at 42 °C for 30 min, and at 85 °C for 5 min.
Subsequently, quantitative real-time PCR was performed with TaqMan MicroRNA assay probes and the TaqMan Universal Master mix (Thermo Fisher) using reverse transcription product. The following thermal cycle protocol was performed using the Rotor-Gene Q real-time PCR cycler (Qiagen, Hilden, Germany): initial denaturation at 95 °C for 2 min followed by 40 cycles of 95 °C for 15 sec and 60 °C for 1 min.
Data were analysed with real-time PCR Rotor Gene Q Software 2 (Thermo Fisher). MiR-125b-fold changes were calculated by the delta Ct method (2^-ΔCt) and its expression was determined using RNU48. All reactions were run in duplicate.