Evaluation of optimal RNA extraction method from human carotid atherosclerotic plaque.
Author(s): Ahmed S, Shaffer A, Geddes T, Studzinski D, Mitton K, Pruetz B, Long G, Shanley C
Publication: Cardiovasc Pathol, 2015, Vol. 24, Page 187-90
PubMed ID: 25534148 PubMed Review Paper? No
Purpose of Paper
This paper compared the quantity and integrity of RNA from matched snap-frozen carotid artery plaque specimens after extraction using three different methods and RNAlater-preserved specimens extracted using Trizol.
Conclusion of Paper
RNA yields were approximately 4.5-fold higher from snap-frozen and RNAlater-preserved carotid plaque specimens when extracted using Trizol rather than using column-based methods (RNeasy), but RNA integrity numbers (RIN) were significantly lower than using RNEasy. The highest integrity RNA was isolated from snap-frozen tissue using RNeasy, with lower RIN values (RIN<7) occurring in specimens with low overall RNA yield (<50 ng/µL).
This study compared RNA yield and integrity in matched snap-frozen carotid artery plaques specimens extracted using three different methods and RNAlater-preserved specimens extracted using Trizol. Atherosclerotic plaque specimens were collected by carotid endarterectomy from an unspecified number of symptomatic patients (i.e., presenting symptoms of transient ischemic attack or stroke) and asymptomatic patients. Each lesion was subdivided into sections (≤ 5 mm) that were either snap-frozen in liquid nitrogen (LN2) and then stored in LN2 or preserved in RNAlater at room temperature for 24 h and then stored at -80°C. RNAlater specimens were homogenized prior to RNA extraction and snap-frozen specimens were pulverized with a mortar and pestle. RNA was extracted using a column-based extraction (RNeasy mini kit), a column-based extraction with proteinase K digestion (RNeasy Fibrous Tissue Mini Kit), phenol-based extraction (Trizol), or a combination of phenol-based processing with column-based extraction (Trizol with RNeasy columns). RNA was quantified spectrophotometrically and analyzed using a bioanalyzer to determine RIN. mRNA levels were assessed by RT-PCR analysis of glyceraldehyde-3’- phosphate dehydrogenase (GAPDH) expression.
Summary of Findings:
RNA extracted using RNeasy from a single RNAlater-preserved specimen revealed slightly higher yields when cholesterol crystal was removed (method not described) than from the whole plaque, and the highest yields were obtained when the whole plaque was digested with proteinase K before RNeasy extraction, but differences were not statistically signiﬁcant (RIN values 0 to 2.7). RNA yields from snap-frozen and RNAlater-preserved specimens from the same tissue were an average of 4.6-fold higher when extracted using Trizol than using RNeasy method or combination of Trizol and RNeasy columns, but RNA integrity was significantly lower (not computed and 6.4, respectively versus 8.6 and 6.9 respectively), GAPDH mRNA levels by real-time PCR were comparable in RNAlater and snap-frozen specimens extracted using Trizol and slightly higher from snap-frozen specimens extracted using RNeasy columns with or without Trizol extraction. The average RIN of 25 additional snap-frozen specimens using RNeasy was 8.7, with only three specimens having a RIN < 7 and low overall RNA yield (<50 ng/µL).
- Coronary Artery Disease
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA Spectrophotometry RNA RT-PCR
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Frozen
Analyte Extraction and Purification Analyte purification RNeasy mini kit
RNeasy Fibrous Tissue Mini Kit
Trizol with RNEasy columns
Storage Storage temperature LN2
Stored 24 h at RT then -80°C