USE OF INTERNAL CONTROLS TO INCREASE QUANTITATIVE CAPABILITIES OF THERIBONUCLEASE PROTECTION ASSAY

Through the use of two internal controls, we have developed an improve d method of quantitating ribonuclease protection assay (RPA) results. A truncated sense RNA fragment and an antisense RNA fragment for the g ene of interest were transcribed from PCR fragments containing T7 bact erial promoters. An 18S ribosomal RNA fragment was also used. When rad iolabeled antisense and 18S probes, along with sense fragment and samp le RNA, were hydridized, digested with RNase A/T1 and gel-electrophore sed, three distinct bands resulted. The antisense RNA fragment bound t o the sense RNA fragment confirmed the integrity of the reaction. The antisense RNA fragment bound to endogenous mRNA measured the amount of specific gene expression in the sample. The 18S RNA fragment bound to endogenous mRNA determined the actual amount of sample added to the g el. Using the specific activities of the antisense and 18S transcripts , and scintillation counts of the protected fragments, we calculated t he amounts of message and total RNA on the gel, determining picogram o f message per microgram of total RNA. Final results were not based on assumed original amounts of RNA placed in the assay nor were they bias ed by lane-to-lane variations. Through the described adaptations, we h ave developed a well-controlled RPA that accurately and reproducibly q uantified gene expression.