Qscript mir cdna synthesis kit

Total microRNA (miRNA) was isolated from different iPSC-EV samples using the miRNeasy Micro Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Reverse transcription was carried out using commercial qScript miR cDNA synthesis kit (Quantabio, Beverly, MA, USA). The PerfeCTa® Universal PCR Primer (QuantaBio) has been designed and validated to work specifically with PerfeCTa SYBR Green SuperMix using miRNA cDNA produced. The levels of miR-133, miR-155, miR-221, and miR-34a were determined. U6, SNORD48, and SNORD44 were examined as housekeeping genes for the normalization of miR expression levels (primer sequences are shown in Table 1). Real-time RT-PCR reactions were performed on an Applied Biosystems Quantstudio 7 flex (Applied Biosystems, Foster City, CA, USA), using SYBR1 Green PCR Master Mix (Applied Biosystems). The amplification reactions were performed as follows: 10 min at 95 °C, and 40 cycles of 95 °C for 15 s and 60 °C for 30 s, and 70 °C for 30 s. Fold variation in gene expression was quantified by means of the comparative Ct method: 2^(−(∆C_(t treatment)−∆C_(t control)), which is based on the comparison of expression of the target gene (normalized to the endogenous control) between the compared samples.

Total microRNA (miRNA) was isolated from different EV samples using the miRNeasy Micro Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Reverse transcription was carried out using the commercial qScript miR cDNA synthesis kit (Quantabio, Beverly, MA, USA). The PerfeCTa^® Universal PCR Primer (QuantaBio, Beverly, MA, USA) has been designed and validated to work specifically with the PerfeCTa SYBR Green SuperMix using miRNA cDNA produced. The levels of miRs were determined with SNORD44 as a housekeeping gene for normalization of miR expression levels (Primer sequences are shown in Supplementary Table S2). Real-time RT-PCR reactions were performed on an Applied Biosystems Quantstudio 7 flex (Applied Biosystems, Foster City, CA, USA), using SYBR1 Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The amplification reactions were performed as follows: 10 min at 95 °C, and 40 cycles of 95 °C for 15 s and 60 °C for 30 s, and 70 °C for 30 s. Fold variation in gene expression was quantified by means of the comparative Ct method: $2^{-(\Delta C_{t treatment}-\Delta C_{t control})}$ , which is based on the comparison of expression of the target gene (normalized to the endogenous control) between the compared samples.