Evaluation of optimal RNA extraction method from human carotid atherosclerotic plaque

Samreen Ahmed, Andrew Shaffer, Timothy Geddes, Diane Studzinski, Kenneth Mitton, Barbara Pruetz, Graham Long, Charles Shanley

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

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.

Original languageEnglish (US)
Article number6818
Pages (from-to)187-190
Number of pages4
JournalCardiovascular Pathology
Volume24
Issue number3
DOIs
StatePublished - May 1 2015
Externally publishedYes

Keywords

  • Atherosclerosis
  • Carotid artery
  • Gene expression
  • RNA
  • Real-time PCR

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