Cfx96 detection system

Extraction of total RNA, separation of cytoplasm and nuclear RNA was performed as described in the previous study [8 (link)]. Real-time PCR analysis of mRNA and circRNA was performed using SYBR Premix Ex TaqTM (Takara, Japan) and Bio-Rad CFX96 detection system (USA). Real-time PCR analysis of miRNA was performed using the All-in-OneTM miRNA qRT-PCR detection kit (GeneCopoeia) and the Bio-Rad CFX96 detection system (USA). Primers for detection were obtained from GENEray (Supplementary Table 1).

Total RNA was extracted using the TaKaRa MiniBEST Plant RNA Extraction Kit (Takara, Dalian, China). RNase-free DNase-I was used to remove any contaminating genomic DNA in the solution. First-strand cDNA synthesis was conducted using a FastQuant RT Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. The qRT-PCR analysis of all 12 ZmSMRs was performed using primers that were designed according to the ZmSMRs’ sequences and using an NCBI Primer-BLAST online instrument (Additional file 11). The amplification products were controlled within a size range of 130 bp to 250 bp (Additional file 11). The maize actin (Zm00001d013873) gene was used as an internal control. All primers were synthesized by Sangon Biotech Company. The qRT-PCR assays were performed in optical 96-well plates with three technical replicates using the BIO-RAD CFX96 Detection System (Bio-Rad, CA, USA). Each reaction was performed in 20 μL of a SuperReal Premix Plus (SYBR Green) reaction mixture (TIANGEN, Beijing, China), following the manufacturer’s instructions. The relative expression ratio of each gene was calculated using the 2^-△△CT method [63 (link)].

miRNA in Exo^Hypoxic and Exo^Normoxic were analyzed using TaqMan^® Array MicroRNA Cards with pre-amplification kit (Life Technologies, Applied Biosystems). 350 ng of miRNA was used as input in each reverse transcription (RT) reaction. The RT reaction and pre-amplification steps were done according to the manufacturer’s instructions. miRNAs were reverse transcribed using TaqMan^® MicroRNA Reverse Transcription Kit (Life Technologies, Applied Biosystems). RT reaction products from the exosome sample were further amplified using the Custom PreAmp Primer Pool. The expression profile of miRNAs was determined using the TaqMan^® Universal Master Mix II (Life Technologies, Applied Biosystems) in an Applied Biosystems 7900HT thermal cycler using the manufacturer’s recommended program. Finally, all the raw data from each array were retrieved from the 7900HT and run on Data Assist Software ver.3.1 (Applied Biosystems). In another set of experiments, we collected exosomes from the conditioned medium of RWPE1, LNCaP, PC-3 and DU145 cells; RNA was isolated from cells and exosomes, followed by miRNA cDNA synthesis (Applied Biological Materials Inc., BC, Canada). Real-time PCR was performed using SYBR Green master mix on a Bio-Rad CFX96 detection system (Bio-Rad, Hercules, CA). The fold change in expression levels was determined using RNU6 and 5S rRNA as an internal control.