Ana Cecília Valadares1,2, Hagen Gorki1, Andreas Liebold1, Markus Hoenicka1
1University of Ulm Medical Center, Department of Cardiothoracic and Vascular Surgery, Ulm, Germany
Background and aim of the study: The isolation of high-quality RNA is an important first step in gene expression studies. However, difficult tissue disruption, low cell content and low RNA content makes consistent RNA extraction from human aortic valve tissue a challenging task.
Methods: A protocol has been developed for the successful isolation of high-quality RNA from human aortic valve samples by optimizing RNA extraction protocols based on a comparison of commercial kits.
Results: Guanidinium thiocyanate-phenolchloroform
extraction was found to be a prerequisite for successful purification. Two protocols based on this extraction were further optimized. RNA quality and quantity were assessed spectrophotometrically, using a Bioanalyzer and by PCR analysis of several housekeeping genes. Optimized parameters included storage in RNAlater™, DNase digestion, the amount of tissue, homogenization time, and freezing of tissue after homogenization.
Conclusion: The modified protocol for fatty and fibrous tissue achieved satisfactory results for gene expression analysis of human aortic valve samples.
The Journal of Heart Valve Disease 2017;26:185-192
|Extraction of Total RNA from Calcified Human Heart Valves for Gene Expression Analysis|
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