Comparison of commercially available kits for exosomal RNA isolation from plasma: qRT-PCR analysis of exosome marker CD9

Abstract

Exosomes, which are released by cells in body fluids such as serum and plasma, are nanovesicles containing RNAs and proteins. In recent years, RNAs or proteins within exosomes have attracted considerable attention because they serve as potential diagnostic biomarkers, especially in cancer. In addition, reagents for extracting exosomes from serum and/or plasma have been developed worldwide, but no definite conclusion on their extraction efficiency has been reached. At present, the conventional ultracentrifugation method still remains more reliable than methods using commercial extraction reagents. However, there are many problems associated with ultracentrifugation, such as the need to prepare the equipment and acquire operation skills, making it unsuitable for efficient exosome research and clinical applications. Thus, we are awaiting a reliable method for extracting exosomes using commercially available reagents. In this study, we compared two different commercial kits, ExoRNeasy (QIAGEN), which has been reported to have a high extraction efficiency in the analysis of proteins and mi-RNA, and the newly developed Plasma/Serum Exosome Purification Kit (NORGEN), which has not yet been analyzed elsewhere. An evaluation assay using healthy human plasma was performed by RNA determination and RT-qPCR analysis of the exosome marker CD9. For both RNA quantification and RT-qPCR analysis of CD9, we obtained reliable mRNA extraction results using ExoRNeasy. Thus, we confirmed the reliable extraction of mRNA in exosomes using commercially available reagents. On the basis of these results, we consider that we should proceed with our research on the establishment of novel biomarkers and clinical applications of these kits.