Miscript sybr green kit

Reverse transcription to cDNA was performed with miScript Reverse Transcriptase Mix (QIAGEN) using 50 ng of RNA for each reaction. Samples were stored at−20°C until qPCR. The miScript SYBR green kit (QIAGEN) was used to run qPCR on a QuantStudio 3 Real-Time PCR System (ThermoFisher Scientific, Waltham, MA). Primers assays for selected miRNAs, miR-32, miR-105a, miR-143, miR-181a-1, and miR-214, were acquired (QIAGEN). Melting curves were evaluated for each run, ensuring all reactions had a single peak on the melting curve and verifying the amplification of a unique PCR product.
The small nuclear RNU6B and three additional miRNAs, miR-152, miR-872, and miR-1842, were evaluated as endogenous reference genes for the normalization of miRNA expression by RT-qPCR. These markers were chosen based on RNA-Seq evaluation for consistent expression throughout control and iUC samples and literature review (26 (link)–29 (link)). The exogenous control miR-39 was also used.

Reverse transcription was performed on 156 ng of total RNA using the miScript II RT Kit (QIAGEN, Valencia, CA, USA) using the HiSpec buffer. Real-time PCR was performed with the miScript SYBR Green Kit (QIAGEN, Valencia, CA, USA). Forward primers (miScript primer assays, QIAGEN, Valencia, CA, USA) designed to detect the following mature miRNAs were used: hsa-let-7i-5p (MS00003157), hsa-miR-10a-5p (MS00031262),, hsa-miR-151b (MS00037513), hsa-miR-16–2-3p (MS00008813), hsa-miR-16-5p (MS00031493), hsa-miR-1910-5p (MS00016464), hsa-miR-423-5p (MS00009681), hsa-miR-92a-3p (MS00006594) and hsa-miR-92b-3p (MS00032144). The reactions were performed in triplicate using the QuantStudio 3 real-time PCR system (USA) with the following conditions: 95 °C for 15 min, 60 amplification cycles of 94 °C for 15 s, 55 °C for 30 s and 70 °C for 30 s. The miRNA expression was normalized using the ∆∆CT method with the recommended housekeeping gene RNU6B (MS00033740). RNU6B expression was consistent in all the samples both within and across experimental conditions. No statistically significant differences (p > 0.05) in the expression of RNU6B between sEVs and/or cells samples measured by Standard Deviation of CT were identified.

In order to quantify mature miRNA transcript levels as well as precursor miRNA levels, the miScript kit was used (Qiagen, Germany). Reverse transcription (RT) was performed using 300–400 ng of total RNA and “HiFlex” RT Buffer, which allows detection of both microRNA and messengerRNA. Temperature settings were chosen according to the suppliers recommendations (37 °C for 1 h, 95 °C for 5 min). cDNA was diluted 1:4 in nuclease-free water and qPCRs were run in quadruplicates using the miScript SYBR Green Kit (Qiagen, Germany) on the Rotorgene Q instrument (Qiagen, Germany): 95 °C → 15 min, 40 cycles of 94 °C → 15 s, 55 °C → 30 s, 70 °C → 30 s. SYBR Green fluorescence was measured at 70 °C and 80 °C. Commercial primer assays (Qiagen, Germany) were used for mature miRNA quantification. In-house designed primer assays were used for precursor-miRNA quantification (primer sequences are listed in Supporting Table 1).