title: Evaluation of optimal RNA extraction method from human carotid atherosclerotic plaque.
abstract: Investigating molecular mechanisms involved in the formation of carotid atherosclerotic plaques has been challenging. Isolating high-quality RNA from plaque tissue can be difficult because of acellularity, calcification, and degradation. It is essential that the mRNA isolated from this tissue preserves and reflects the actual relative gene expression. Two common methods for RNA preservation, snap-freezing and stabilizing reagent, were compared using surgically resected human carotid atherosclerotic tissue. In addition, isolation methods were compared for integrity and quantity: column-based extraction, phenol-based extraction, and a combination of the two. We found that using a stabilizing reagent with column filtration resulted in the lowest yield and quality. Phenol-based extraction resulted in higher yields but also increased fragmentation. Snap-frozen tissue coupled with column-based extraction yielded the highest quality. The higher quality and quantity RNA obtained when processing snap-frozen tissue with column-based extraction make it possible to use difficult sample types for molecular downstream applications.
authors.affiliation: Beaumont BioBank, Beaumont Health System, Royal Oak, MI, USA. Electronic address: Samreen.Ahmed@beaumont.edu.
authors.forename: Samreen
authors.initials: S
authors.lastname: Ahmed
authors.affiliation: Department of Surgery, Beaumont Health System, Royal Oak, MI, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Shaffer
authors.affiliation: Beaumont BioBank, Beaumont Health System, Royal Oak, MI, USA.
authors.forename: Timothy
authors.initials: T
authors.lastname: Geddes
authors.affiliation: Department of Surgery, Beaumont Health System, Royal Oak, MI, USA.
authors.forename: Diane
authors.initials: D
authors.lastname: Studzinski
authors.affiliation: Eye Research Institute, Control of Gene Expression Laboratory & the Pediatric Retinal Research Laboratory, Oakland University, Rochester, MI, USA.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Mitton
authors.affiliation: Beaumont BioBank, Beaumont Health System, Royal Oak, MI, USA.
authors.forename: Barbara
authors.initials: B
authors.lastname: Pruetz
authors.affiliation: Department of Surgery, Beaumont Health System, Royal Oak, MI, USA.
authors.forename: Graham
authors.initials: G
authors.lastname: Long
authors.affiliation: Department of Surgery, Beaumont Health System, Royal Oak, MI, USA; Oakland University William Beaumont School of Medicine, Rochester, MI, USA; Wayne State University School of Medicine, Detroit, MI, USA.
authors.forename: Charles
authors.initials: C
authors.lastname: Shanley
mesh_terms: D002340:Carotid Artery Diseases / Q000235:genetics*; D006801:Humans; D058226:Plaque, Atherosclerotic / Q000235:genetics*; D012313:RNA / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction; D014021:Tissue Preservation / Q000379:methods*
chemical_list: D012313:RNA
keywords: Atherosclerosis; Carotid artery; Gene expression; RNA; Real-time PCR