Per-unit-living tissue normalization of real-time RT-PCR data in ischemic rat hearts

Area of Science:

• Molecular Biology
• Cardiovascular Research
• Biotechnology

Background:

• Accurate gene expression analysis in acute ischemic heart tissue requires stable internal reference genes to account for dead cells.
• Previous studies have not adequately addressed the issue of reference gene stability in this context.

Purpose of the Study:

• To investigate the expression stability of seven internal reference genes in Langendorff-perfused rat hearts subjected to ischemia-reperfusion.
• To identify reliable reference genes for accurate real-time RT-PCR data normalization in ischemic heart studies.

Main Methods:

• Langendorff-perfused rat hearts were subjected to ischemia-reperfusion.
• Expression levels of seven internal reference genes (cyclophilin A, GAPDH, RPL-32, PolR2A, β-actin, 18S rRNA) were analyzed.
• Gene expression stability was assessed per unit of living tissue and per unit of total tissue weight.

Main Results:

• Cyclophilin A, GAPDH, RPL-32, and PolR2A mRNA showed stable expression per unit of living tissue, with GAPDH exhibiting the highest stability (R = 0.11).
• GAPDH mRNA degradation correlated with infarct size and RNA quality (R = 0.90).
• β-actin and 18S rRNA showed stable expression per unit of tissue weight but correlated positively with infarct size, leading to normalization errors.

Conclusions:

• GAPDH is a highly reliable reference gene for normalizing real-time RT-PCR data in ischemic heart tissue.
• Geometric averaging of carefully selected reference genes is recommended for normalization.
• 18S rRNA is unsuitable for normalization due to altered mRNA/rRNA ratios in ischemic tissues.