Enhanced sensitivity by optimizing biotin incorporation into RNA probe. (A) EtBr-stained denaturing formaldehyde–MOPS agarose gel of Nluc 5′-UTR RNA probes that were transcribed in vitro using the indicated ratios of biotinylated UTP (bioU) to unlabeled UTP. Total UTP concentration was the same in all reactions. Assuming equal incorporation of bioU and U, we estimate this probe will contain 10% biotinylated nucleotides at the 1:2 bioU:U ratio and 21% biotinylated nucleotides at the 3:1 bioU:U ratio. (B) Chemi-Northern detection was performed on identical blots using Nluc 5′-UTR probe with the indicated ratio of bioU:U. The gels were analyzed and imaged under identical conditions using the same exposure setting. 7SL RNA was detected using a biotinylated DNA oligo probe on the same blots, as a loading control. (C) Quantitation of the Chemi-Northern signals in panel B. Signal intensity for Nluc in each lane was normalized to its corresponding 7SL RNA signal. Fold change was then calculated relative to the mean value of the 1:2 bioU:U probe. The mean, SD, and the three data points for each condition are shown in the graph.
