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Technique / Molecular Biology / RNA transcriptional post-transcriptional regulation / RNase protection assay
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Ribonuclease protection assay. (Curr Protoc Mol Biol. 2001 May;Chapter 4:Unit4.7.) Gilman M. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA. Sequence-specific hybridization probes of high specific activity are prepared by cloning the probe sequence downstream of a bacteriophage promoter. The plasmid is cleaved with a restriction enzyme, and the plasmid DNA is transcribed with bacteriophage RNA polymerase, which efficiently transcribes the cloned sequence into a discrete RNA species of known specific activity and high abundance. The RNA is purified by removal of the DNA template, protein, and the unincorporated label. Alternatively, the probe is purified by gel electrophoresis, as described in a support protocol. The probe RNA is hybridized to sample RNAs and the hybridization reactions are treated with ribonuclease to remove free probe, leaving intact fragments of probe annealed to homologous sequences in the sample RNA. These fragments are analyzed by electrophoresis on a sequencing gel and the presence of the target mRNA is revealed by the appearance of an appropriately sized fragment of the probe. Introduction to Ribonuclease Protection Assays (RPA) and RPA procedure Ribonuclease Protection Assay (RPA assay) 
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Last update: 
16-May-2008 03:42 pm
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The IP10 (CXCL10) specific cDNA probe of the mCK-5c multiprobe RNase protection assay kit carries two nucleotide insertions that complicate the interpretation of results. Cytokine. 2008 Feb;41(2):182-6
Authors: Sauder C, Pedras-Vasconcelos J, Puig M, Verthelyi D
RNase protection assays (RPA) employing multiprobe sets are powerful tools to simultaneously measure transcription of several different genes. We used BD Biosciences/Pharmingen's mouse chemokine probeset mCK-5c to measure chemokine gene expression in brain and spleen tissue of mice. Depending on the RPA protocol used, we observed differences in the relative amounts of transcripts for interferon-inducible protein 10 (IP-10) and T-cell activation-3 (TCA-3). Isolation and sequencing of the IP-10 specific gene from the mCK-5c probeset revealed two nucleotide insertions in the probe that are not present in the natural IP-10 cDNA. We show that these insertions cause RNase A-dependent degradation of the protected IP-10 mRNA yielding a fragment indistinguishable in size from that specific for TCA-3, thus leading to over-interpretation of TCA-3 expression as well as underestimation of IP-10 gene expression levels.
RNase protection assay for quantifying gene expression levels. Methods Mol Biol. 2007;366:145-58
Authors: Qu Y, Boutjdir M
Quantifying the level of mRNA is central to the study of mammalian gene expression. Conventional approaches such as Northern blotting are often prone to low sensitivity and reproducibility. The RNase protection assay (RPA) provides a sensitive alternative for the detection and quantification of mRNA. The RPA is based on the hybridization in solution of a labeled single-stranded antisense RNA probe with a target mRNA. After hybridization, single-strand specific RNases are then used to digest away unhybridized RNA. The hybrid can be resolved by a denaturing gel. Subsequent detection will reveal the appropriate-sized gel band corresponding to the target mRNA. The major advantage of RPA is the high sensitivity and the simultaneous detection and quantification of multiple mRNA targets in a single RNA sample. The primary limitation of RPA is the lack of information on transcript size.
RNase protection assay. Methods Mol Biol. 2007;362:343-8
Authors: Emery P
The RNase protection assay is a standard approach to determine mRNA levels of a gene of interest in different tissues, developmental stages, or times of the day. Splicing or promoter variants can be studied with specific probes. It is widely used in chronobiology to study the temporal profile of expression of circadian genes and the effects of genetic manipulation on these oscillations. Methods to generate the riboprobes and to perform the RNase protection assay itself are described in this chapter.
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