Life taqman advanced mirna cdna synthesis kit

The same amount of Caenorhabditis elegans cel-39-3p miRNA was spiked into each sEVs sample as an external calibration for RNA extraction, reverse transcription, and miRNA amplification. Total RNA was extracted and purified from plasma sEVs using miRNeasy® Mini kit (Qiagen, cat. 217,004). TaqMan™ advanced miRNA assays were performed for miRNA quantification using Life Taq Man Advanced miRNA cDNA Synthesis Kit (Life Tech, Carlsbad, CA, cat.A28007) and Life Taq Man Fast Advanced Master Mix (Life Tech, Carlsbad, CA, cat.4444557). miRNA quantification was obtained with qPCR using specific miRNA TaqMan gene expression probes (Life Tech, Carlsbad, CA) and an ABI7500 qPCR system (Applied Biosystems) with a miRNA-specific probe mix and cDNA template according to the manufacturer’s protocols. The following probes were used: 478,221 for hsa-let-7b-3p, 478,721 for hsa-miR-150-3p, 477,915 for hsa-miR-145-3p, 477,906 for hsa-miR-139-3p, 477,975 for hsa-miR-21-5p, 479,448 for hsa-miR-30a-5p (Life Technologies). All samples were normalized by the initial biofluid input volume used for RNA extraction, and calibrated by the spike-in cel-39-3p to eliminate the minute bias caused by different RNA isolation efficiencies and PCR efficiencies among samples.