RNase Protection Assay Analysis of mRNA for TGFβ1(-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, TGFβ(2), and TGFβ(3) 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.