Protection assay

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-... 

Cytokine profiling has also been measured as a function of changes in cytokine mRNA expression using either reverse transcription polymerase chain reaction (RT-PCR) [87, 91-93] or ribonuclease protection assay (RPA) [94-97], Measurement of cytokine transcripts by RT-PCR revealed that prolonged exposure to TMA induced increased levels of IL-4 mRNA expression compared with treatment with DNCB [87,92-93]. However, expression of the type 1 cytokine IFN-y by DNCB-activated LNC was variable and failed to discriminate between contact and respiratory allergens [87,91,93). A similar profile was observed for freshly isolated tissue analyzed by RPA. This somewhat less... 

Plitnik, L.M., et al., Cytokine profiling for chemical sensitizers Application of the ribo-nuclease protection assay and effect of dose. Toxicol. Appl. Pharmacol., 179, 145, 2002. 

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). 

Theoretical rate of modification of an introduced cysteine using four different concentrations of MTS (10 xM, 20 ulM, 30 ii.M, and 60 ulM, ). The pseudo first-order rate constants (kfl are 0.1285, 0.2445,0.4849, and 0.7564, respectively. When corrected for the concentration-dependence of MTS modification, the second-order rate constants (k2) are 12,900, 12,275 12,122 and 12,606 M s respectively. Although all k2 values are similar, the early phase of modification (<20 s) is less well described when higher concentrations (>20 xM) of the reagents are applied (see inset). Based on these data, we would use 10 xM for control experiments and protection assays... 

The protection assay is carried out in much the same way as the control rate, the primary difference being that an agonist/antagonist/modulator is coapphed with the MTS reagent. The purpose of this kind of experiment is to determine if any such hgand will alter the rate of reaction. 

Foster On the basis of the protection assay I showed I would say that we have no evidence for melanopsin outside the eye. But it might be in the brain or pineal at low levels. We have preliminary evidence that there may be different forms of melanopsin in the teleosts, and that they may be expressed in different sites. 

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. 

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... 

T.M. and Rimsza, L.M. (2007) Quantitative nuclease protection assay in paraffin-embedded tissue replicates prognostic microarray gene expression in diffuse large-B-cell lymphoma. Lab. Invest.,... 

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