A RELIABLE EXTERNAL CONTROL FOR RIBONUCLEASE PROTECTION ASSAYS

A method is described for generating an external spiked human RNA cont rol to enhance the reliability of assessment of gene expression in tum our extracts. Spiking with an external standard RNA controls for all s ubsequent steps of analysis on a lane by lane basis and allows for uni form comparison of the gene of interest as a fraction of total RNA, pa rticularly when multiple samples are not available. The antisense prob e that is being used to detect endogenous gene expression is also used as an external control. A sense riboprobe is made from the same vecto r. Because of the flanking RNA polymerase sites incorporated in both p robes, hybridization with the sense riboprobe at a much lower concentr ation than the antisense probe generates a larger product that can be readily separated from the endogenous protected fragment. This method is generally applicable to any riboprobe that has a T3 and T7 RNA poly merase site and allows any externally added riboprobe use for assessin g endogenous gene expression to be used as the external spike central.