Quantification of biotinylated RNA probes for in situ hybridization using chemiluminescence

For reliable detection of mRNA by non-radioactive in situ hybridization, calibration and standardization of the individual steps involved are essential. We describe a method that allows determination of the size and integrity as well as quantification of biotinylated RNA probes in a single experiment. Serial dilutions of biotinylated RNA probes generated by promoter-mediated in vitro transcription were size-separated by gel electrophoresis in the presence of known amounts of 5'-biotinylated oligomers which served as internal standard. Following immobilization onto nylon membranes and visualization by chemiluminescence, optical densities of probes and internal standards were measured by densitometry and analysed by linear regression. RNA probes complementary to the human homeobox genes HOX-C6, -C8 and -C9 were quantified. Four different 5'-biotinylated oligomers (20, 35, 50 and 75 bases) were tested as internal standards. Concerning the separation of probe and oligomer in the gel, transfer properties and efficiency of binding to the membrane, the oligomer of 35 bases was found to be the best internal standard with highest reproducibility. Comparison of probe concentration by spectrophotometry and the described method showed a good correlation, indicating that our method is a reliable assay for quantitative and qualitative control of biotin-labelled probes.