A Comparison of Various “Housekeeping” Probes for Northern Analysis of Normal and Osteoarthritic Articular Cartilage RNA

Several approaches are commonly used to normalize variations in RNA loading on Northern blots, including: ethidium bromide (EthBr) fluorescence of 18S or 28S rRNA or auto-radiograms of radioactive probes hybridized with constitutively expressed RNAs such as elongation factor-la (ELF), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), actin, 18S or 28S rRNA, or others. However, in osteoarthritis (OA) the amount of total RNA changes significantly and none of these RNAs has been clearly demonstrated to be expressed at a constant level, so it is unclear if any of these approaches can be used reliably for normalizing RNA extracted from osteoarthritic cartilage. Total RNA was extracted from normal and osteoarthritic cartilage and assessed by EthBr fluorescence. RNA was then transferred to a nylon membrane hybridized with radioactive probes for ELF, G3PDH, Max, actin, and an oligo-dT probe. The autoradiographic signal across the six lanes of a gel was quantified by scanning densitometry. When compared on the bas...