Purification of cardiac myocytes from human heart biopsies for gene expression analysis.
The collection of gene expression data from human heart biopsies is important for understanding the cellular mechanisms of arrhythmias and diseases such as cardiac hypertrophy and heart failure. Many clinical and basic research laboratories conduct gene expression analysis using RNA from whole cardiac biopsies. This allows for the analysis of global changes in gene expression in areas of the heart, while eliminating the need for more complex and technically difficult single-cell isolation procedures (such as flow cytometry, laser capture microdissection, etc.) that require expensive equipment and specialized training. The abundance of fibroblasts and other cell types in whole biopsies, however, can complicate gene expression analysis and the interpretation of results. Therefore, we have designed a technique to quickly and easily purify cardiac myocytes from whole cardiac biopsies for RNA extraction. Human heart tissue samples were collected, and our purification method was compared with the standard nonpurification method. Cell imaging using acridine orange staining of the purified sample demonstrated that >98% of total RNA was contained within identifiable cardiac myocytes. Real-time RT-PCR was performed comparing nonpurified and purified samples for the expression of troponin T (myocyte marker), vimentin (fibroblast marker), and alpha-smooth muscle actin (smooth muscle marker). Troponin T expression was significantly increased, and vimentin and alpha-smooth muscle actin were significantly decreased in the purified sample (n = 8; P < 0.05). Extracted RNA was analyzed during each step of the purification, and no significant degradation occurred. These results demonstrate that this isolation method yields a more purified cardiac myocyte RNA sample suitable for downstream applications, such as real-time RT-PCR, and allows for more accurate gene expression changes in cardiac myocytes from heart biopsies.
Kosloski
L M
LM
University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108, USA.
Bales
I K
IK
Allen
K B
KB
Walker
B L
BL
Borkon
A M
AM
Stuart
R S
RS
Pak
A F
AF
Wacker
M J
MJ
D000199:Actins / Q000235:genetics; D015415:Biomarkers; D001706:Biopsy; D002469:Cell Separation / Q000379:methods*; D006331:Heart Diseases / Q000235:genetics* / Q000473:pathology*; D006801:Humans; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000502:physiology*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D012313:RNA / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D020107:Troponin T / Q000235:genetics; D014746:Vimentin / Q000235:genetics
D000199:Actins; D015415:Biomarkers; D020107:Troponin T; D014746:Vimentin; D012313:RNA