1243. Title: Clotomics: The Clot Genome Atlas
Authors: Dominic A. Nistal; YoneJung Yoon; Colin Lamb, BA; Rui Song, BA; Christopher Kellner, MD (New York, NY)
In the past three years, mechanical thrombectomy for has proven to be an effective adjunct to t-PA in multiple randomized clinical trials for the treatment of acute ischemic stroke (AIS). There is evidence that AIS clot biology is active with cellular processes that could potentially be interrupted to sensitize the clot to medication or be studied to better understand stroke etiology or pathophysiology. In this study, we describe a novel method for isolating protein and RNA from ischemic stroke thrombi in order to perform multi-omic analysis.
Seven cerebrovascular thrombi were prospectively collected after subjects underwent thrombectomy for AIS. During the procedure, clots were placed into RNAlater (Invitrogen) stabilization product at room temperature (RT). To validate RNAlater preservation in RT, protein and RNA were isolated from mouse brains at one of four time points (fresh, 24, 48, and 72 hours) remaining at RT until isolation procedures were performed using the Invitrogen PARIS kit. The quantity and quality of RNA and protein from the clots were measured using the Nanodrop one/oneC. Protein expression levels were determined using the Proseek multiplex immunoassay, inflammatory panel (http://www.olink.com/). RNA sequencing was performed using the NovaSeq 6000 with 150 PE Sequencing S4 throughput.
Mean protein and RNA concentration were 41.8 mg/mL and 55.97 ng/uL with an average A260/A280 ratio of 0.40 and 2.13 respectively. There was no significant difference of the quality or concentration of RNA extracted at different time points (p=0.1305).
In this study, we propose a novel protocol for isolating and analyzing protein and RNA from acute ischemic stroke thrombi without the need for freezing the samples or performing extraction on fresh tissue. Multi-omic analysis of acute ischemic stroke clots will allow for the development of personalized diagnostic, treatment, and recovery models for patients.