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Isolation of RNA from Acute Ischemic Stroke Clots Retrieved by Mechanical Thrombectomy
Vincent M Tutino1,2,3,4, Sarah Fricano1,2, Kirsten Frauens1,3
1Canon Stroke and Vascular Research Center, University at Buffalo, Buffalo, NY 14203, USA.
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.
