Abstract

Circulating microvesicles (cMV) are lipid-encapsulated bodies that are secreted from various tissues and can be detected in a number of body fluids, including plasma. Once collected from plasma, they can be exploited diagnostically for their protein and RNA signatures. Mutations in KRAS are diagnostically important for predicting response to chemotherapy and prognosis. A blood-based method of assessing KRAS mutation status would be helpful for patients with colorectal cancer (CRC). Traditional methods of KRAS detecting examine the genomic DNA sequence. We developed a method to sequence exon 2 of KRAS mRNA. The limit of KRAS mutation detection was 0.78 ug of mutation-positive exomes per mL of plasma for both Pyrosequencing and Sanger sequencing. To further enhance mutation detection, we sorted cMV from CRC patients by first capturing cMV with a CRC-associated membrane protein and then sorting for CD63 positive events. A Taqman gene expression assay was not sensitive enough to detect KRAS in the sorted samples. However, Pyrosequencing was sufficient to identify mutant and wild-type sequences in patient plasma samples.