Quantitative analysis of mRNA

Baugh LR, Hill AA, Brown EL et al. Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 2001 29 E29. 

Al-Mahrouki, A. A., Krylov, S. N. (2005). Calibration-free quantitative analysis of mRNA. Anal Chem 77, 8027-8030. 

Buck KJ, Harris RA, Sikela JM. 1991. A general method for quantitative PCR analysis of mRNA levels for members of gene families application to GABAa receptor subunits. Biotechniques 11 636-641. 

As stated earlier, activation of endothelial cells by pro-inflammatory stimuli leads to the expression of cell adhesion molecules and cytokines such as IL-6 and IL-8. The expression and hence modulation of surface expressed adhesion molecules by e.g. targeted delivery of inhibitors of NFkB, can be measured using flow cytometric analysis or whole cell ELISA techniques. Cytokine production can be measured in the supernatant of cultured cells or in biological fluids. Furthermore, competitive or quantitative RT-PCR analysis of mRNA levels of cell adhesion molecules or cytokines, allows the transcriptional activity of the genes of interest to be estimated. 

Lauriat T. L., Dracheva S., Chin B., Schmeidler J., Mclnnes L. A., and Haioutunian V. (2006). Quantitative analysis of glutamate transporter mRNA expression in prefrontal and primary visual cortex in normal and schizophrenic brain. Neuroscience 137 843-851. 

Zhang, J. and Cashman, J. R. (2006) Quantitative analysis of FMO gene mRNA levels in human tissues. Drug Metab. Dispos. 34 (1), 19-26. 

The mRNA level of a bioprocess-relevant gene can be used as an early measure of its expression status. A simple but labor-intensive offline technique for the mRNA analysis of one or a limited number of genes is Northern blotting. Following the separation of the RNA sample in a polyacrylamide gel, the RNA is blotted on a membrane and detected by the hybridization of labeled sequence-specific probes. This technique does not only allow a quantitative analysis of the mRNA level but also gives information about the length and potential degradation products of the RNA. 

The field of gene expression analysis has only recently become of broader interest to users of mass spectrometry. The reason for this lag in interest was simply the lack of methods allowing the absolute quantification of NAs in general, or mRNA in particular. Mass spectrometry only allows an endpoint analysis, and even then a standard for normahzation (such as a second allele) is required. Hence, the approaches used have focused on the semi-quantitative analysis of allelic expression which, as described above, is a rather narrow-albeit expanding-field of research. 

Gilliand G, Perrin S, Blanchard K, Bunn HF. 1990b. Analysis of cytokine mRNA and DNA detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad... 

We found that it is necessary to run several sets of differential display primers prior to an analysis of the distribution of differential display bands. This allows for a comparison between different independent reactions using different PCR primers to assess the quality of individual cDNA samples and discriminate between sample-to-sample variability and potential positive bands that are consistently found in different repUcates. The presence or absence of a specific band in lanes corresponding to independent experimental samples indicates a reproducible difference in the relative amount of cDNA in a given sample, which should reflect differences in mRNA levels. However, the interpretation of the differential display results is not always straightforward. For example, a thick band can reflect quantitative differences in the initial concentration of a specific cDNA between samples or can represent comigration of two bands. Replication of the PCR reactions for samples that have differences in banding pattern will eliminate a significant number of false positive differential display differences. Also, in some cases, it may be informative to alter the electrophoresis conditions to maximize resolution of a band of interest prior to isolation, reamplification, and further analysis of potential positive bands. 

This multiprobe RPA offers the advantages of its sensitivity and capacity to simultaneously quantitate several mRNA species in a single sample of total RNA. This allows comparative analysis of different mRNA species within samples, moreover, by incorporating probes for housekeeping gene transcripts, the levels of individual mRNA species can be compared between samples. Furthermore, the assay is highly specific and quantitative owing to the RNase sensitivity of mismatched base pairs and the use of solution-phase hybridization driven toward completion by excess probe. Finally, the multiprobe RPA can be performed on total RNA preparations derived by standard methods from either frozen tissues or cultured cells. 

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