Isolation of RNA from Acute Ischemic Stroke Clots Retrieved by Mechanical Thrombectomy

Abstract

Mechanical thrombectomy (MT) for large vessel acute ischemic stroke (AIS) has enabled biologic analyses of resected clots. While clot histology has been well-studied, little is known about gene expression within the tissue, which could shed light on stroke pathophysiology. In this methodological study, we develop a pipeline for obtaining useful RNA from AIS clots. A total of 73 clot samples retrieved by MT were collected and stored in RNALater and in 10% phosphate-buffered formalin. RNA was extracted from all samples using a modified Chemagen magnetic bead extraction protocol on the PerkinElmer Chemagic 360. RNA was interrogated by UV–Vis absorption and electrophoretic quality control analysis. All samples with sufficient volume underwent traditional qPCR analysis and samples with sufficient RNA quality were subjected to next-generation RNA sequencing on the Illumina NovaSeq platform. Whole blood RNA samples from three patients were used as controls, and H&E-stained histological sections of the clots were used to assess clot cellular makeup. Isolated mRNA was eluted into a volume of 140 µL and had a concentration ranging from 0.01 ng/µL to 46 ng/µL. Most mRNA samples were partially degraded, with RNA integrity numbers ranging from 0 to 9.5. The majority of samples (71/73) underwent qPCR analysis, which showed linear relationships between the expression of three housekeeping genes (GAPDH, GPI, and HPRT1) across all samples. Of these, 48 samples were used for RNA sequencing, which had moderate quality based on MultiQC evaluation (on average, ~35 M reads were sequenced). Analysis of clot histology showed that more acellular samples yielded RNA of lower quantity and quality. We obtained useful mRNA from AIS clot samples stored in RNALater. qPCR analysis could be performed in almost all cases, while sequencing data could only be performed in approximately two-thirds of the samples. Acellular clots tended to have lower RNA quantity and quality.

Gene expression profiles of ischemic stroke clots retrieved by mechanical thrombectomy are associated with disease etiology

“…Most individuals had a middle cerebral artery or internal carotid artery occlusion (57.9% and 28.9%, respectively). For all clot samples we analyzed, RNA quality and sequencing metrics have been previously reported 10. In brief, the 38 RNA samples had an average concentration of 22.5 ng/µL, an average 260/280 of 2.1, and average RNA integrity number of 3.3 (samples without quantifiable RNA integrity numbers were assessed via manual inspection of the electrophoresis gel).…”

“…The isolation and sequencing analysis of AIS clot RNA is described in detail in our previous paper 10. In brief, clot RNA was isolated via a modified magnetic bead capture protocol for the PerkinElmer Chemagic 360, and eluted into a final volume of 140 μL.…”

“…Recently, a protocol for the isolation of AIS clot RNA for downstream quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and RNA sequencing (RNAseq) applications has been published 10. Here, we followed this protocol and analyzed the transcriptomes of clots collected from patients with AIS undergoing MT.…”

New Insights into Cerebral Vessel Disease Landscapes at Single-Cell Resolution: Pathogenetic and Therapeutic Perspectives

“…The study of the cellular landscape of atherosclerosis often entails handling of limited clinical tissue samples such as artery biopsies, atherosclerotic plaques, or thrombi, making recovery of sufficient quantities of vital cells difficult. In particular, the cellular components of thrombi are very scarce, limiting RNA studies of mechanical thrombectomy [ 24 ]. RNA-seq methods for investigating small vessel disease, atherosclerosis, and thrombosis have been described in detail previously [ 22 , 25 , 26 ].…”

“…In this surgery, the intima is collected together with the plaque, with the tunica media left in place. None of these studies were performed from material obtained during endovascular thrombectomy procedures, which may yield lower quality material with fewer cells because thrombi consist mainly of platelets and clotting factors, as bulk RNA-seq analysis has shown [ 24 ]. Careful tissue conservation from collection to analysis is crucial.…”