RNase Protection Assay Analysis of mRNA for TGF1-3 in Ovarian Tumors

RNase protection assays provide a level of sensitivity some 20-50-fold greater than Northern blots, and can be used to accurately identify and quantify different mRNA species within gene families even when a high degree of sequence homology exists. Sequence homology between TGFβ1, TGFβ2 , and TGFβ3 mRNAs is approximately 70%. Identification of these subspecies by Northern is often complicated by cross-hybridization of probes. In the RNase protection assay, detection of mRNA relies on the formation of a RNA:RNA hybrid of absolute specificity. Single-stranded RNA is digested with a single-strand specific RNase mixture and even single basepair mismatches will lead to loss of hybridization signal. Therefore, this technique provides a readily quantifiable means of detecting multiple RNA species, either individually or simultaneously. This technique has been applied to the study of both ovarian carcinoma cell lines (1 ) and tumors (2 ) yielding valuable insights into the role of TGFβs in ovarian carcinomas.