Total exosome rna kit

Briefly, cellular RNA was isolated with TRIzol reagent (Invitrogen), while exosomal miRNA was extracted using the Total Exosome RNA Kit (Ambion) and MirVana RNA isolation kit (Ambion) according to recommendations. GAPDH and U6 served as an endogenous control to normalize the expression level of miR-22-3p and VE-cadherin, respectively. The 2^−ΔΔCt method [23 (link)] was utilized to evaluate relative expression levels. The primer sequences were designed as follows: miR-22-3p Forward: 5′-AAGCUGCCGUUGAAGAACUGU-3′; Reverse: 5′-GTGCAGGGTCCGAGGT-3′; U6 Forward: 5′-CTCGCTTCGGCAGCACA-3′; Reverse: 5′-AACGCTTCACGAATTTGCGT-3′; VE-cadherin Forward: 5′-CCGCTCGAGACCAATTCCTATAACCTTC-3′; Reverse: 5′-TATGCGGCCGCTTCCCCATGAGGCTCTCTG-3′; GAPDH Forward: 5′-CAGGAGGCATTGCTGATGAT-3′; Reverse: 5′-GAAGGCTGGGGCTCATTT-3′.

miR-146a is a type of miRNA associated with immunity, participating in cell differentiation, cell proliferation, cell immune response, and release of inflammatory mediators [15 (link)]. Exosomal miRNA was extracted using the Total Exosome RNA Kit (Ambion) and MirVana RNA isolation kit (Ambion) following the manufacturer's instructions. U6 was used as the internal reference for qualification of the cellular miRNA. The PCR primers were purchased from Biomics Biotechnologies (Nantong, China).

Briefly, cellular RNA and zebrafish RNA were isolated with TRIzol reagent (Invitrogen), while exosomal miRNA was extracted using the Total Exosome RNA Kit (Ambion) and MirVana RNA isolation kit (Ambion) according to recommendation. U6 was used as the corresponding internal reference for qualification of the cellular miRNA. The synthetic spike control (cel-miR-39) served as an invariant control for qualification of the exosomal miRNA.