Developmental Changes in Total Cardiac mRNA: RNA Complicates Interpretation of Reference Genes

Abstract

Finding genes that do not vary with age for use as reference or “housekeeping” can be difficult, as can understanding what experimental gene normalization means in a developmental context. We analyzed expression levels of several potential reference genes in the developing sheep heart, as well as global myocardial mRNA levels compared to total nucleic acid levels. Total nucleic acid, total RNA, messenger RNA (mRNA), and individual gene levels were determined by spectroscopy, dot blot, and PCR. Results were analyzed by ANOVA followed by Tukey’s test, and results reported at P<0.05. Potential reference genes hyaluronidase 2, beta‐2‐microglobulin and ribosomal protein L37a were all found to vary by developmental age. The ribosomal RNA 18s did not vary with age. While total RNA relative to total nucleic acid was constant across development, mRNA was a greater fraction of total RNA in the late‐term fetal heart. These results demonstrate of the difficulty of interpreting normalization of myocardial mRNA expression levels across development.Support or Funding InformationR01HL142483