Type II collagen expression in small, biopsy-sized samples of cartilage using a new method of RNA extraction.
The extraction of mRNA from cartilage samples is complicated by the presence of proteoglycans and the low cellular density of the tissue. We required a method that would enable mRNA to be extracted from small biopsy-sized samples of cartilage. The method had to produce consistent results and sufficient RNA for Northern and PCR analysis. Methods of total RNA extraction, previously shown to be effective for cartilage, were compared with a new technique in which an oligo (dT) conjugated to biotin hybridises to the mRNA. The hybrids are captured with covalently coupled streptavidin paramagnetic particles. Samples of growth plate cartilage, including those specifically from the upper (proliferative and transitional) and lower (fully hypertrophic) zones, were collected and some were frozen at -70 degrees C. Samples for extraction by the paramagnetic method weighed approximately 80 mg and approximately 12 micrograms of mRNA was extracted from fresh tissue samples. The yield from similar frozen samples of the same weight was about a seventh of that from the fresh tissues. 5 micrograms of the mRNA from each sample was run on a gel, and a Northern blot was prepared and probed with a [32P]-labelled antisense RNA probe to type II collagen cDNA. A distinct band of type II collagen mRNA was detected (5.3 Kb) in the samples from the upper (proliferative and transitional) zone. The traditional methods of extracting RNA from cartilage required far greater quantities of tissue and the RNA produced was frequently degraded. The results obtained using the paramagnetic bead method precluded further trials with modification of the traditional methods of mRNA extraction.(ABSTRACT TRUNCATED AT 250 WORDS)
Thorp
B H
BH
AFRC Roslin Institute (Edinburgh), Roslin, Midlothian, U.K.
Armstrong
D G
DG
Hogg
C O
CO
Alexander
I
I
D000818:Animals; D001706:Biopsy; D015152:Blotting, Northern; D002356:Cartilage / Q000378:metabolism* / Q000473:pathology; D002645:Chickens; D003094:Collagen / Q000235:genetics* / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008280:Magnetics; D008863:Microspheres; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012313:RNA / Q000032:analysis*; D015203:Reproducibility of Results
D012313:RNA; D003094:Collagen