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

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 stu...

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Autores principales: Tutino, Vincent M., Fricano, Sarah, Frauens, Kirsten, Patel, Tatsat R., Monteiro, Andre, Rai, Hamid H., Waqas, Muhammad, Chaves, Lee, Poppenberg, Kerry E., Siddiqui, Adnan H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8536169/
https://www.ncbi.nlm.nih.gov/pubmed/34681010
http://dx.doi.org/10.3390/genes12101617
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author Tutino, Vincent M.
Fricano, Sarah
Frauens, Kirsten
Patel, Tatsat R.
Monteiro, Andre
Rai, Hamid H.
Waqas, Muhammad
Chaves, Lee
Poppenberg, Kerry E.
Siddiqui, Adnan H.
author_facet Tutino, Vincent M.
Fricano, Sarah
Frauens, Kirsten
Patel, Tatsat R.
Monteiro, Andre
Rai, Hamid H.
Waqas, Muhammad
Chaves, Lee
Poppenberg, Kerry E.
Siddiqui, Adnan H.
author_sort Tutino, Vincent M.
collection PubMed
description 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.
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spelling pubmed-85361692021-10-23 Isolation of RNA from Acute Ischemic Stroke Clots Retrieved by Mechanical Thrombectomy Tutino, Vincent M. Fricano, Sarah Frauens, Kirsten Patel, Tatsat R. Monteiro, Andre Rai, Hamid H. Waqas, Muhammad Chaves, Lee Poppenberg, Kerry E. Siddiqui, Adnan H. Genes (Basel) Article 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. MDPI 2021-10-14 /pmc/articles/PMC8536169/ /pubmed/34681010 http://dx.doi.org/10.3390/genes12101617 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Tutino, Vincent M.
Fricano, Sarah
Frauens, Kirsten
Patel, Tatsat R.
Monteiro, Andre
Rai, Hamid H.
Waqas, Muhammad
Chaves, Lee
Poppenberg, Kerry E.
Siddiqui, Adnan H.
Isolation of RNA from Acute Ischemic Stroke Clots Retrieved by Mechanical Thrombectomy
title Isolation of RNA from Acute Ischemic Stroke Clots Retrieved by Mechanical Thrombectomy
title_full Isolation of RNA from Acute Ischemic Stroke Clots Retrieved by Mechanical Thrombectomy
title_fullStr Isolation of RNA from Acute Ischemic Stroke Clots Retrieved by Mechanical Thrombectomy
title_full_unstemmed Isolation of RNA from Acute Ischemic Stroke Clots Retrieved by Mechanical Thrombectomy
title_short Isolation of RNA from Acute Ischemic Stroke Clots Retrieved by Mechanical Thrombectomy
title_sort isolation of rna from acute ischemic stroke clots retrieved by mechanical thrombectomy
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8536169/
https://www.ncbi.nlm.nih.gov/pubmed/34681010
http://dx.doi.org/10.3390/genes12101617
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