MRNA expression, measurement

The former hypothesis has been evaluated using mRNA expression measurements in human tissues and it was found that ABCB1 is generally expressed at higher levels with the 3435C [83, 93-95]. These observations were replicated with cotransfection of equal amounts of plasmid and it was concluded that the 3435T allele... 

It is hard to predict the future of mRNA expression measurement. What we can be sure of is that proteomics will gain ground from genomics as increasing amounts of data on expression levels of mature proteins become available. By the same token, mRNA expression measurement will probably continue to have its applications, e.g. in diagnostics, especially since genomics experiments are and can be expected to remain cheap and easy compared with proteomics experiments. 

Friedman et al. (2000) tested this approach on a data set for yeast cell cycle expression patterns provided by Spellman and co-workers (1998), writing that This data set contains 76 gene expression measurements of the mRNA levels of 6177 S. cerevisiae ORFs. These experiments measure six... 

Harrison DC et al. The use of quantitative RT-PCR to measure mRNA expression in a rat model of focal ischemia -caspase-3 as a case study. Brain Res Mol Brain Res 2000 75 143-149. 

As pDNA and mRNA transfection differ in both the timing of mRNA expression and the gross amount of mRNA delivered to the cell, it is important to identify a suitable time point to measure miR-mediated repression. We observe that at any time point after transfection, pDNA transfections have higher measurable levels of miR-mediated repression compared to mRNA transfections (Fig. 6.2C). This difference may, in part, reflect a time lag of active miR-protein-complex formation relative to the onset of translation of the transfected Renilla luciferase mRNA. For single time point experiments, we decided to measure miR-mediated repression in mRNA and DNA transfections at 16 and 24 h, respectively. 

Pisa, E.K. et al., OKT3-induced cytokine mRNA expression in human peripheral blood mononuclear cells measured by polymerase chain reaction, ScandL J. Immunol., 36, 745, 1992. 

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... 

Techniques that measure mRNA expression and ligand binding assays that measure receptor expression have been used extensively to identify those nAChR subtypes that are expressed in dopamine neurons. In situ hybridization studies using mouse (Marks et al. 1992 Grady et al. 1997) and rat (Le Novere et al. 1996) brain have detected the mRNAs for all of the known nAChR subunits, except a2 and p4, in... 

A number of studies have repeatedly measured increased CRF-like immunoreactivity in the CSF of untreated patients with major depression (e.g., Nemeroff et ah, 1984). A recent study using serial CSF sampling over 30 hours has provided evidence for inadequately high CRF activity in major depression in the face of sustained hypercortisolism (Wong et ah, 2000). Postmortem studies have further provided evidence for increased CRF concentrations and CRF mRNA expression in hypothalamic tissue of depressed patients as well as decreased CRF receptor binding, likely due to chronic CRF hypersecretion, in the frontal cortex of suicide victims (Nemeroff et ah, 1988 Raadsheer et ah, 1994, 1995). These findings are consistent with indices of increased CRF activity in the hypothalamus and other structures in animals models of early-life stress. Direct measures of central CRF release in humans with early-life stress are still unavailable. 

The investigators measured messenger ribonucleic acid (mRNA) expression levels of MDRl and CYP3A4 in mucosal cells of the upper jejunum collected during living-donor liver transplantation in 48 recipients. Tacrolimus was initiated at an oral dose of 0.075 mg/kg every 12 hours and adjusted on the basis of trough levels in whole blood. 

Another important issue is what can be used to predict change in protein levels. In a review of the literature, it is apparent from the measurements in yeast and mouse liver that there is yet no strong correlation between mRNA level and that of proteins, especially in the case of poorly expressed genes. Therefore, conducting gene expression measurements may not be sufficient to infer protein expression. Thus, the analytical methods used in proteomics must provide detection of proteomes present in multiple-modified forms at relatively low levels. 

Figure 8.4 Pomegranate juice reduces oxidative stress in lesions and in macrophages in vivo and in vitro studies. Mean ( SD) of the effect of PJ consumption on lipid peroxides in human carotid lesions (A) and in mouse peritoneal macrophages (B). J774 A1 macrophages were incubated with increasing concentrations of PJ for 90 minutes at 37°C. (C) Cellular oxidative stress measured as DCFH oxidation, (D) PON2 lactonase activity, and (E) PON2 mRNA expression. = p < 0.01 (aftervs. before PJ consumption in humans, and PJ vs. control in mice, and incubation in vitro with PJ vs. control without PJ).

The effect of nicotine, the main principle of tobacco, on PKC activity was measured as a function of time. At a concentration of 100 nmol/1, nicotine caused an increase in PKC activity in endothelial cells from human adult CNS. The increase in PKC activity was significant in 30 s and attained maximum levels at 2 min. In order to assess the significance of the nicotine-induced PKC activation in the observed increase in plasminogen activator inhibitor-1 (PAI-1) production, the effect of nicotine was measured in the presence of the PKC inhibitor GF-109203-X. In these conditions, nicotine had no effect on PAI-1 mRNA levels. Similar results were obtained with another PKC inhibitor (calphostin C), demonstrating that in CNS-endothelial cells PAI-1 mRNA expression and protein production are dependent on the activation of PKC [31]. 

Fig. 3. Expression of MDR-l, MRP, and p2-microglobulin mRNAs in drug resistant SW620 Ad20 and 300 cells. The three panels demonstrate the dilution series from which mRNA expression levels can be calculated. Dilution yields PCR products which are two-fold different when measured by densitometry from cDNA diluted to provide the exponential range. Serial dilutions in the exponential range for MRP and [T-microglobulin are shown in the next two panels.

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