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.

Effects of muscle type, castration, age, and compensatory growth rate on androgen receptor mRNA expression in bovine skeletal muscle.

“…To normalize AR data for differences in total RNA input, the risk for which increases by lowering initial RNA quantity in the system, the 18S rRNA content was determined. This ribosomal RNA fraction is relatively constant in total RNA (Davies et al, 1997). Assuming a nonlinear relationship with the AR mRNA level, we opted for a regression-based approach with 18S rRNA as a covariable to remove variation in AR mRNA levels explained by differences in 18S rRNA.…”

Knocking down the diencephalic thyrotropin-releasing hormone precursor gene normalizes obesity-induced hypertension in the rat

“…Essentially, 10 g of total diencefalic RNA were hybridized with 1 ϫ 10 5 cpm of each labeled antisense RNA probe, treated with ribonucleases A and T1, and purified by proteinase K and phenol-cloroform-isoamyl alcohol (24:24:1), following the procedure of Davis et al (5). The protected probes were resuspended in 5 l of loading buffer (80% formamide, 1 mM EDTA, pH 8.0, 0.1% bromophenol blue, 0.1% xylene cyanol) and fractionated on a 6% polyacrylamide containing 8 mol/l urea gel.…”

Reverse protection assay: a tool to analyze transcriptional rates from individual promoters

“…The RPA method has been modified to serve for different tasks, such as measuring the radioactive signals by scintillation counting [RiPPA method; [2]], and replacing radiolabeled with biotinylated probes [3]. Adding constitutively transcribed or in vitro synthesized RNA as an internal standard during RNA isolation allows quantification of the RNA analyzed by RPA [4,5]. …”