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 constitutively expressed RNAs such as elongation factor-1alpha (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 basis of total RNA, the coefficient of variation was lowest for 28S ethidium bromide fluorescence and oligo-dT (approximately 7%), followed by 18S ethidium bromide fluorescence and G3PDH (approximately 13%). When these values were normalized to DNA concentration, the coefficient of variation exceeded 50% for all signals. Total RNA and the signals for 18S, 28S rRNA, and oligo-dT all correlated highly. These data indicate that osteoarthritic chondrocytes express similar ratios 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 RNA. All of these "housekeeping" probes are expressed at greater levels by osteoarthritic chondrocytes when compared with normal chondrocytes. Thus, while G3PDH is satisfactory for evaluating the amount of RNA loaded, its level of expression is not the same in normal and osteoarthritic chondrocytes.
Matyas
J R
JR
Department of Anatomy and Pathology, McCaig Centre for Joint Injury and Arthritis Research, University af Calgary Health Sciences Centre, Alberta, Canada. jmatyas@acs.ucalgary.ca
Huang
D
D
Adams
M E
ME
D000199:Actins / Q000235:genetics; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D015152:Blotting, Northern / Q000379:methods / Q000592:standards*; D002358:Cartilage, Articular / Q000473:pathology; D004247:DNA / Q000032:analysis; D004268:DNA-Binding Proteins / Q000235:genetics; D004285:Dogs; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D010003:Osteoarthritis / Q000235:genetics* / Q000473:pathology; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D012313:RNA / Q000032:analysis*; D015347:RNA Probes; D012337:RNA, Ribosomal, 18S / Q000032:analysis; D012339:RNA, Ribosomal, 28S / Q000032:analysis; D014157:Transcription Factors
D000199:Actins; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C499330:MAX protein, human; C067850:Myc associated factor X; D020648:Peptide Elongation Factor 1; D015347:RNA Probes; D012337:RNA, Ribosomal, 18S; D012339:RNA, Ribosomal, 28S; D014157:Transcription Factors; C068422:Max protein, mouse; D012313:RNA; D004247:DNA