A Critical Assessment of the RNAse Protection Assay as a Means of Determining Exon Sizes

• Published in: Analytical biochemistry Vol. 209; no. 2; pp. 360 - 366
• Main Authors: Lau, E.T., Kong, R.Y.C., Cheah, K.S.E.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.03.1993; Elsevier
• Subjects: Analytical biochemistry: general aspects, technics, instrumentation; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Biological and medical sciences; Exons; Fundamental and applied biological sciences. Psychology; Humans; Kinetics; Mice; Molecular Sequence Data; Neurofilament Proteins - genetics; Neurofilament Proteins - isolation & purification; Ribonucleases - chemistry; RNA Probes; RNA, Messenger - analysis; Sensitivity and Specificity; Molecular hybridization; Transcription; Gel electrophoresis; Transcription promoter; Gene expression; Plasmid; Exon; Messenger RNA; Gene; Collagen; Concentration effect; Enzymatic digestion; Nucleotide; Cleavage; Molecular probe; Protection
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1993.1135

The RNAse protection assay is a highly sensitive assay which is commonly used to detect specific hybridization between complementary RNAs and to determine exon sizes in gene characterization studies. Unfortunately, each of the numerous steps involved in the assay could give artifacts depending on the probe used. In this study, common causes of artifacts have been identified using riboprobes which identify exons of known sizes. The RNAse concentration and duration of digestion used were found to be critical factors affecting exon size estimations. Five different riboprobes were tested to obtain a consensus optimum RNAse condition-10 μg/ml RNAse A, 0.5 μg/ml RNAse T1-enabling the correct determination of exon sizes. This condition was further analyzed for its specificity when RNAse protection assays were performed between highly homologous RNA fragments from two different species. Results show that this concentration of RNAse would efficiently cleave a minimum of two nucleotide mismatches. Single nucleotide mismatches were frequently not cleaved by the same RNAse concentration making it possible to detect the correct exon size regardless of such sequence polymorphisms in gene sequences.