Themircute plus mirna first strand cdna synthesis kit

The extraction of total RNA from exosomes and reverse transcription into cDNA were performed with a themiRcute Plus miRNA First‐Strand cDNA Synthesis Kit (#KR211‐02, Tiangen Biotech Co., Ltd), according to the manufacturer's recommendation. The miRNA RT‐qPCR analysis was performed with a miRcute Plus miRNA qPCR Detection Kit (SYBR Green) (#FP411‐02, Tiangen Biotech Co., Ltd) and a Bio‐Rad CFX384 TouchTM instrument (Bio‐Rad). Total RNA from cells was isolated using the TRIzol reagent according to the manufacturer's instructions (Invitrogen), and cDNA was synthesized using the PrimeScript^TM RT Reagent Kit with gDNA Eraser (TaKaRa). The RT‐PCR was subsequently determined using a TransStart Top Green qPCR SuperMix (Transgen, China). RT‐qPCR was performed to measure the expression levels of exosomal miRNAs and YWHAZ with chicken 5S RNA and GAPDH mRNA used as internal controls. Data were analysed using the 2^−ΔΔCt method. The primers of miRNA, mRNA and 5s RNA are presented in Table S1.

The expression of miRNAs was confirmed using the stem-loop qRT-PCR method. Total RNA was extracted by TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendation (Invitrogen, USA) and then reverse-transcribed into cDNA using a Reverse Transcriptase M-MLV (TaKaRa) and Hairpin-itTM microRNA qPCR Quantitation Kit (GenePharma, Shanghai, China). qPCR was performed using a SYBR^® Select Master Mix kit and standard protocols on a Step One Plus Real-Time PCR System (Applied Biosystems, USA). For let-7 miRNAs and RNA sequencing data validation, the Poly(A) Plus real-time PCR method was used. Total RNA was isolated and reverse-transcribed into cDNA by a themiRcute Plus miRNA First-Strand cDNA Synthesis Kit (#KR211-02, Tiangen Biotech Co., Ltd, Beijing, China) and subsequently determined using a miRcute Plus miRNA qPCR Detection Kit (SYBR Green) (#FP411-02, Tiangen Biotech Co., Ltd, Beijing, China) according to the manufacturer’s recommendation. U6 small nuclear RNA was used as an internal control for miRNAs, and mRNA levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The 2^−ΔΔCt comparative method was used to analyze the expression levels. The mRNA, miRNA and U6 primers are listed in Supplemental Table 1.