QUANTIFICATION OF BIOTINYLATED RNA PROBES FOR IN-SITU HYBRIDIZATION USING CHEMILUMINESCENCE

For reliable detection of mRNA by non-radioactive in situ hybridizatio n, calibration and standardization of the individual steps involved ar e essential. We describe a method that allows determination of the siz e and integrity as well as quantification of biotinylated RNA probes i n a single experiment. Serial dilutions of biotinylated RNA probes gen erated by promotor-mediated in vitro transcription were size-separated by gel electrophoresis in the presence of known amounts of 5'-biotiny lated oligomers which served as internal standard. Following immobiliz ation onto nylon membranes and visualization by chemiluminescence, opt ical densities of probes and internal standards were measured by densi tometry and analysed by linear regression. RNA probes complementary to the human homeobox genes HOX-CG, -C8 and -C9 were quantified. Four di fferent 5'-biotinylated oligomers (20, 35, 50 and 75 bases) were teste d as internal standards. Concerning the separation of probe and oligom er in the gel, transfer properties and efficiency of binding to the me mbrane, the oligomer of 35 bases was found to be the best internal sta ndard with highest reproducibility. Comparison of probe concentration by spectrophotometry and the described method showed a good correlatio n, indicating that our method is a reliable assay for quantitative and qualitative control of biotin-labelled probes.