Hybridization-protection assay

Acridinium ester—labeled chemiluminescent probes have been utilized to detect the specific protein-coding transcripts and to distinguish between transcripts that code for the 190-kDa protein and the two closely related 210-kDa proteins. The assay is called the hybridization protection assay (D3). In this assay, RNA isolated from the patient s white blood cells is first amplified by PCR. The amplified product is incubated with the chemiluminescent probe. The unhybridized probe is removed by selective hydrolysis in sodium tetraborate buffer, containing surfactant Triton X-100 at pH 8.5, in an incubation step at 60°C for 6 min. After the sample is cooled to room temperature, the chemiluminescence of the hybridized probe is measured in a luminometer. The procedure is reported to detect one leukemic cell in a population of a million or more normal cells. It is also rapid, requiring less than 30 min. Its reliability has been attested to by correlation with results obtained on karyotypic and Southern blot analysis (D3). 

D3. Dhingra, K., Talpaz, M., Riggs, M. G., Eastman, P. S., Zipf, T., et al.. Hybridization protection assay A rapid, sensitive, and specific method for detection of Philadelphia chromosome-positive leukemias. Blood 77, 238-242 (1991). 

Hybridization protection assay with acridinium ester probes... 

A luminescent probe detection system called the hybridization protection assay, or HP A, makes use of an acridinium ester-derivatized oligonucleotide that is hybridized to the amplified DNA (02). Unhybridized probe is preferentially... 

The second technique uses a homogeneous assay format with no physical separation between hybridized and nonhybridized probes. The method is the result of a discovery that for a suitably positioned acridinium ester, within an oligonucleotide sequence, hybridization prevents hydrolytic attack on the ester by hydroxide ions. Such hydrolysis would lead to the inactivation of the chemiluminescent label (see Section 3.2.1). This differential susceptibility to hydrolysis forms the basis for a so-called hybridization-protection assay (HPA), which is schematically illustrated in Fig. 30. 

Fig. 30. Schematic illustration of the hybridization protection assay (HPA). The acridinium ester is covalently attached to a DNA probe. Upon hydrolysis of the phenyl ester group, there is no possibility of forming a dioxetaneone intermediate and hence the reaction is dark i.e., there is no light emission. By contrast, chemiluminescence of the hybridized probe (shown in the lower part of the figure) is minimally affected. Adapted from Nelson et at. (N6), with permission.

Another class of readout measures RNA expression levels, with the three most common methods being chip-based hybridization/fluorescence techniques, realtime polymerase chain reaction (RT-PCR) and quantitative nuclease protection assays (QNPA) [48, 49]. Chip-based methods are widely used for whole-genome scans (discussed in more detail below), but have a disadvantage that they are relatively expensive and so are not really high throughput. The quantitative reproducibility and dynamic range of these chip-based methods are also lower than for the other RNA readout techniques. RT-PCR is a more quantitative technique for measuring transcript levels, and is typically run for up to 40 transcripts at a time. QNPA is another... 

RNase protection assay. This is a sensitive method to determine (1) the amount of a specific mRNA present in a complex mixture of mRNA and/ or (2) the sizes of exons which comprise the mRNA of interest. A radioactive DNA or RNA probe (in excess) is allowed to hybridize with a sample of mRNA (for example, total mRNA isolated from tissue), after which the mixture is digested with... 

Logical extensions of this method are the SI nuclease protection assay (Reyes and Wallace, 1987) and the simultaneous quantification of several mRNA species by solution hybridization with oligonucleotides (Section 12.3) (O Donovan et al., 1991). 

RNase protection assays (RPA) are based on the property of RNase to digest ii RNA, but not ds RNA, and its principles and applications resemble those of SI analysis (Lynn et al., 1983 Zinn et at, 1983) (Fig. 12.3). The sequence of interest is inserted in a plasmid downstream of a bacteriophage promoter (e.g., pUC118, pT7, etc.. Table 4.4). The purified plasmid is then restricted downstream of the inserted DNA and the linearized plasmid is transcribed in the presence of a labeled rNTP precursor. The transcript should be complementary to the RNA to be studied and an excess of probe is hybridized to its target. Any RNA remaining ss is then digested by one or more RNases. The size of the RNase-resistant probe and the... 

Fig. 1. (A) In situ localization of CNTFRa mRNA in coronal section of adult mouse brain. Shown is a low-power dark-field photomicrograph of emulsion-dipped slides of mouse sections hybridized with CNTFRa antisense " S-labeled RNA probe. (B) List of reported distribution of the CNTFRa and method of assessment. Detection of CNTFRa mRNA by Northern Blot, RNase Protection assay or RT-PCR (NRT) or in situ hybridization (ISH). Detection of CNTFRa protein by Western Blot (WB) or immunohistochemistry (IHC). Detection in in vitro primary cell culture (1°).

FGF-8 mRNA is present from E9 to El2 of murine gestation as shown by RNase protection assay and in situ hybridization studies (Heikin-heimo et al., 1994 Ohuchi et al., 1994 Crossley and Martin, 1995). FGF-8 is widely expressed in the telencephalon, diencephalon, and metencepha-lon, particularly in the midbrain-hindbrain junction, and in the developing pituitary gland. These observations suggest an important role of FGF-8 in early brain development. 

Related mRNAs encoding various proteins can be detected by different types of in situ hybridization. For this method, the number of specific mRNAs detectable per tumor sample is limited. However, the advantage of in situ hybridization is the same as in immunohistochemistry where the morphology of the tumor is still visible. Specific mRNA-species can be detected by northern blot, nuclease protection assay or reverse transcription (RT) combined with polymerase chain reaction (PCR). Using the modern real-time PCR protocols, reliable quantification of PCR targets is possible. A more complex approach is possible by using the micro-array technology, where hundreds or even more of mRNAs can be detected simultaneously in a semi-quantitative fashion. 

Figure 8.12 Detection of thrombin by an aptamer-based exonuclease protection assay. Thrombin is binding to its specific aptamer in solution. The free aptamer is hydrolyzed by exonuclease I, whereas the bound aptamer remains intact. The intact aptamer is then hybridized with two connector oligonucleotides and the hybridization product is ligated by DNA ligase. The hgation product is used as a template for amplification by PCR. [From (Wang et al., 2004).]...

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