Use of Internal Controls to Increase Quantitative Capabilities of the Ribonuclease Protection Assay

Abstract

Through the use of two internal controls, we have developed an improved method of quantitating ribonuclease protection assay (RPA) results. A truncated sense RNA fragment and an antisense RNA fragment for the gene of interest were transcribed from PCR fragments containing T7 bacterial promoters. An 18S ribosomal RNA fragment was also used. When radiolabeled antisense and 18S probes, along with sense fragment and sample RNA, were hybridized, digested with RNase A/T1 and gel-electrophoresed, three distinct bands resulted. The antisense RNA fragment bound to 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 gel. Using the specific activities of the antisense and 18S transcripts, and scintillation counts of the protected fragments, we calculated the amounts of message and total RNA on the gel, determining picogram of message per microgram of total RNA. Final results were not based on assumed original amounts of RNA placed in the assay nor were they biased by lane-to-lane variations. Through the described adaptations, we have developed a well-controlled RPA that accurately and reproducibly quantifies gene expression.