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 autoradiograms of radioactive probes hybridized with consti tutively expressed RNAs such as elongation factor-1 alpha (ELF), glyceralde hyde-3-phosphate dehydrogenase (G3PDH), actin, 18S or 28S rRNA, or others. However, in osteoarthritis (OA) the amount of total RNA changes significant ly 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 r eliably 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 b y scanning densitometry. When compared on the basis of total RNA, the coeff icient of variation was lowest for 28S ethidium bromide fluorescence and ol igo-dT (approximate to 7%), followed by 18S ethidium bromide fluorescence a nd G3PDH (approximate to 13%). When these values were normalized to DNA con centration, the coefficient of variation exceeded 50% for all signals. Tota l RNA and the signals for 18S, 28S rRNA, and oligo-dT all correlated highly . These data indicate that osteoarthritic chondrocytes express similar rati os of mRNA to rRNA and mRNA to total RNA as do normal chondrocytes. Of all the "housekeeping" probes, G3PDH correlated best with the measurements of R NA. All of these "housekeeping" probes are expressed at greater levels by o steoarthritic chondrocytes when compared with normal chondrocytes. Thus, wh ile G3PDH is satisfactory for evaluating the amount of RNA loaded, its leve l of expression is not the same in normal and osteoarthritic chondrocytes.