Abstract
BackgroundRecently, some genomic mutations in exosomal DNA have been found to be related to disease progress and clinical outcomes of patients in several cancers. Unfortunately, the methods for exosome isolation and exosomal DNA analysis are still lack of relevant research to ensure their optimal performance and the comparability. Here we aim to establish a protocol for cancer-related mutation detection on exosomal DNA in clinical application.MethodsTaking KRAS mutation in pancreatic cancer as an example, we tested whether the types of blood samples, the potential factors in the courses of exosome isolation and exosomal DNA preparation, as well as the detail in mutation detection by droplet digital PCR (ddPCR) could influence the exosomal DNA analysis.ResultsWe found that the concentration of exosomal DNA from serum was higher than that from plasma, whereas the mutant allele fraction (MAF) of KRAS in serum-derived exosomal DNA was obviously lower. The membrane-based method for exosome isolation showed no evident difference in both exosomal DNA yield and KRAS MAF from the classical ultracentrifugation method. DNase I pretreatment on exosomes could remove the wild-type DNA outside of exosomes and increase the KRAS MAF. PBS might interfere with the effect of DNase I and should not be recommended as resuspension buffer for exosomes if the subsequent experiments would be done with exosomal DNA. Besides, the denaturation of exosomal DNA before droplet generation during ddPCR could effectively improve the total KRAS copy number and mutation-positive droplet number.ConclusionThis study provides some methodological evidences for the selection of the optimal experimental conditions in exosomal DNA analysis. We also suggest a protocol for mutation detection on exosomal DNA, which might be suitable for the clinical testing and could be helpful to the comparison of results from different laboratories.
Highlights
As the current gold standard for cancer diagnosis, tissue biopsy is usually not convenient, for the patients who cannot undergo surgical resection
When we use the membrane-based method for exosome isolation, the manufacturer normally provides Buffer XE as the resuspension buffer for exosomes
Considering the reports that the exosomes obtained through the existing isolation methods might contain some impurities, such as the potential contamination of external DNA associated with the outer membrane of exosomes [18], we tried to remove the external DNA with DNase I treatment before proceeding with the extraction of exosomal DNA
Summary
As the current gold standard for cancer diagnosis, tissue biopsy is usually not convenient, for the patients who cannot undergo surgical resection. In contrast with tissue biopsy, the detection on circulating DNA is minimally invasive, and may better reflect the overall and real-time tumor burden in cancer patients [2,3,4]. The exosomal DNA may exhibit higher molecular weight in comparison with circulating cell-free DNA (cfDNA) [8]. Some tumor-related mutations in exosomal DNA were found to be able to reflect the disease progress and prognosis in patients with pancreatic cancer (PC), colorectal cancer, and so on [9,10,11]. Some genomic mutations in exosomal DNA have been found to be related to disease progress and clinical outcomes of patients in several cancers. We aim to establish a protocol for cancer-related mutation detection on exosomal DNA in clinical application
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