RNase protection

In general, quantitative gene expression results obtained with microarray analyses correspond well to results obtained using other methods.11 However, quantitative data from microarrays are typically less reliable for genes that are very highly or very poorly expressed.12 In addition, microarrays are much less sensitive than real time PCR and RNAse protection assays.13 However, the ability to analyze most of the mRNA spe-... 

Sassone-Corsi In the experiment you showed it is clear that in all those receptors there is extensive alternative splicing. In an RNAse protection assay, picking the probe is crucial. 

Van Gelder Mike Young s group validated their targets by Northern blot or RNAse protection. Yet those stiU don t show up in your list. 

Fig. 4. Changes in A, GS mRNAs and B, GS isoenzymes, during nodule development. The abundances of the mRNAs were measured by an RNase protection technique and the isoenzymes following separation by IEX-HPLC (adapted from Cullimore et al., 1990). GSs=GS synthetase activity. The isoenzymes are as defined in Fig. 3.

Figure 5.8 Detection of PNA-induced 7-globin gene expression in K562 cells with RNase Protection Assay. Lane 1, K562 cells lane 2 to lane 4, K562 cells treated with PNAs for two, three, and four days, respectively lane 5, K562 cells treated with hemin (75 pM) for three days.

The methods used for the evaluation of regulation of gene expression are too numerous to be described in detail here. They include Northern analysis to determine levels of a particular mRNA, nuclear run on to determine whether an increase in mRNA is due to an increase in the rate of transcription, and promoter deletion analysis to identify specific elements in the promoter region responsible for the control of expression. Of much current interest is the use of microarrays that permit the study of the expression of hundreds to thousands of genes at the same time. Reverse transcriptase-polymerase chain reaction and RNase protection assay techniques are used to amplify and quantitate mRNAs, while the electrophoretic mobility shift assay is used to measure binding of a transcription factor to its specific DNA consensus sequence. 

Downstream application [PCR (polymerase chain reaction), cloning, labeling, blotting, RT (reverse transcriptase)-PCR, cDNA synthesis, RNAse protection assays, gene therapy, etc.]... 

Murthy K, Shen SH, Banville D. A sensitive method for detection of mutations—a PCR-based RNase protection assay. DNA Cell Biol 1995 14(1) 87-94. 

DOR gene spans 32 kb from transcription initiation sites located between 140 bp and 390 bp upstream of the ATG translation start codon to a polyadeny-lation site located 1.2 kb downstream of the TGA stop codon. RNase protection analysis of the 5 ends of mouse brain poly(A) + RNA and NG108-15 total RNA resulted in identical patterns of multiple protected fragments, suggesting that the DOR gene is transcribed from multiple initiation sites in the TATA-less, 80% G + C-rich sequence between 140 and 390... 

Figure 2 Nucleotide sequence of DOR gene 5 flanking region. Sequence is numbered relative to + 1 representing the A nucleotide in the ATG translation start codon, indicated by bold type. Positions of minor transcription initiation sites determined from estimates of RNase protected fragment sizes are indicated with bullets ( ) The two strongest transcription start sites are indicated by asterisks ( ) at positions —142 and —324. The cis elements and corresponding trans factors that have been identified are underlined and labeled above the sequence, respectively. (From Ref. 16.)...

Assay using relative light units produced by luciferase as the read out by the method described for DNA transfection reporter assay in Subheading 3.3.5. Other methods of choice to determine gene knockdown are Northern blots, RT-PCR, RNase protection, and branched DNA assays, apart from which assays to measure target protein produced can also be used. 

Several rapid and sensitive methods have been developed to detect mutations in cDNA and genomic DNA. These include ribonuclease (RNase) protection analysis, denaturing gradient gel electrophoresis, and single-strand conformation polymorphism analysis. These methods can be used in conjunction with the PCR technique to detect small deletions or insertions and single base substitutions. 

RNase protection is used to detect the RNase-sensitive site in a synthetic RNA probe that has been hybridized with the DNA fragment to be examined. If a particular mutation lies within the DNA fragment, then the RNA probe is mismatched at the site of the mutation where RNase digestion occurs. 

Wang T, Brown MJ (1999) mRNA quantification by real time TaqMan polymerase chain reaction validation and comparison with RNase protection. Anal Biochem 269 198-201... 

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