Sybr premix ex taq qrt pcr assays

Exosome microRNAs were sequenced in Young-Exo and Aged-Exo. Differentially expressed microRNAs were identified by log₂ |(Fold Change)| > 1 and P < 0.05 with the threshold set for up and down regulated microRNAs.
Total RNA was isolated by TRIzol (Life technologies, United States). RNA concentration was quantitated by the Nano Drop ND-2000 Spectrophotometer (Nano Drop Technologies, Wilmington, DE, United States) and cDNAs were synthesized using PrimeScript^TM RT reagent kit (TaKaRa, Japan). Real-time polymerase chain reaction (PCR) was performed by SYBR Premix Ex Taq qRT–PCR assays (TaKaRa, Japan) with microRNAs, Cel-mir-39, and U6 specific primers (Genscript, Nanjing, China) under 7900HT Real-Time PCR Detection System (Thermo Fisher Scientific, United States). Cel-mir-39 (exosomal) and U6 (cellular) served as external or internal standard to normalize the miRNA expression level using 2^–ΔΔCt method. Primer sequences were listed in Supplementary Table S1.

Mice were euthanized 14 days after PPE infusion. The total RNA of aortae was extracted using the TRIzol reagent (TaKaRa, Japan), according to the manufacturer’s instruction. A measure of 500 nanograms of total RNA was used for reverse transcription using a PrimeScript RT Reagent Kit (TaKaRa). Quantitative RT-PCR on a real-time PCR system (Applied Biosystems) was performed using SYBR Premix Ex Taq qRT-PCR assays (TaKaRa). The Ct value of mRNA was normalized to GAPDH, and the fold change was calculated using the ΔΔCt method. The primer sequences for genes are listed as follows: ACTB, forward primer: GTG​CTA​TGT​TGC​TCT​AGA​CTT​CG and reverse primer: ATG​CCA​CAG​GAT​TCC​ATA​CC; NOS2, forward primer: GTT​CTC​AGC​CCA​ACA​ATA​CAA​GA and reverse primer: GTG​GAC​GGG​TCG​ATG​TCA​C; CCL2, forward primer: TAA​AAA​CCT​GGA​TCG​GAA​CCA​AA and reverse primer: GCA​TTA​GCT​TCA​CAT​TTA​CGG​GT; IFN-γ, forward primer: CAG​CAA​CAG​CAA​GGC​GAA​AAA​GG and reverse primer: TTT​CCG​CTT​CCT​GAG​GCT​GGA​T; IL-1β, forward primer: GAA​ATG​CCA​CCT​TTT​GAC​AGT​G and reverse primer: TGG​ATG​CTC​TCA​TCA​GGA​CAG.