High quality RNA is the key to producing meaningful gene expression analyses. Human cardiac tissue specimens are extremely valuable, but may not always be obtained under optimal conditions and are frequently fibrotic. We provide a practical guide to assist in assessing the efficacy of two different RNA extraction methods applied to these challenging specimens. We describe how to compare ‘single-step’ and ‘multi-step’ extraction processes and discuss how to interpret information available through microfluidic and spectroscopic analyses to evaluate sample quality.