Quantitative transcript analysis

Nguyen, T.T., Nguyen, T.H., Maischberger, T., et al. (2011) Quantitative transcript analysis of the indudble expression system pSlP comparison of the overexpression of Lactobacillus spp. beta-galactosidases in Lactobacillus plan-tarum. Microb Cell Eact 10, 46. 

Lin Z, Crockett DK, Jenson SD, Lim MS, Elenitoba-Johnson KS. Quantitative proteomic and transcriptional analysis of the response to the p38 mitogen-activated protein kinase inhibitor SB203580 in transformed follicular lymphoma cells. Mol Cell Proteomics 2004 3(8) 820-833. 

Quantitative protein analysis is accomplished by combining protein separation, most commonly by high-resolution two-dimensional polyacrylamide gel electrophoresis (PAGE), with MS-(mass spectrometry) based or tandem MS (MSZMS)-based sequence identification of selected, separated protein species. This method is sequential, labor intensive and difficult to automate. It selects against specific classes of proteins, such as membrane proteins, very lar ge and small proteins, and extremely acidic or basic proteins. Tlie technique has a bias toward highly abundant proteins, as lower abundance regulatory proteins (e.g., transcription factors and protein kinases) are rarely detected when total-cell lysates are analyzed. 

Ma, M., Liu, L. Z. Quantitative transcription dynamic analysis reveals candidate genes and key regulators for ethanol tolerance in Saccharomyces cerevisiae. BMC Microbiol. 2010,10,169. 

Lee, J.Y., Pajarillo, E.A., Kim, M.J., Chae, J.P. and Kang, D.K. (2013) Proteomic and transcriptional analysis of Lactobacillus johnsonu PFOl during bile salt exposure by iTRAQ shotgun proteomics and quantitative RT-PCR. J Proteome Res 12, 432-443. 

Because the templates compete for amplification and, in the case of reverse transcription PCR (RT-PCR), also for reverse transcription, any variable affecting amplification has the same effect on both. Thus, the ratio of PCR products reflects the ratio of the initial amounts of the two templates as demonstrated by the function C/W=C (l+ )"/Wi(l+ )n, where Cand Ware the amounts of competitor and wild-type product, respectively, and C and W are the initial amounts of competitor and wild-type template, respectively, (Clementi etal., 1993). From this linear relationship, it could be concluded that a single concentration of competitor could be sufficient for quantitating unknown amounts of wild-type templates. However, in practice, the precise analysis of two template species in very different amounts has proved difficult and cPCRs using three to four competitor concentrations within the expected range of wild-type template concentrations are usually performed. In a recent study of different standardization concepts in quantitative RT-PCR assays, coamplification on a single concentration of a competitor with wild-type template was comparable to using multiple competitor concentrations and was much easier to perform (Haberhausen et al, 1998). 

Godfrey TE, Kim S-H, Chavira M, et al. Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5 nuclease quantitative reverse transcription-polymerase chain reaction. J. Mol. Diagn. 2000 2 84-91. 

ISHII, M., HASHIMOTO, S.I., TSUTSUMI, S., WADA, Y., MATSUSHIMA, K., KODAMA, T., ABURATANI, H., Direct comparison of genechip and SAGE on the quantitative accuracy in transcript profiling analysis, Genome., 2000, 2, 136-143. 

Chawla MK, Lin G, Olson K, Vazdarjanova A, Burke SN, et al. 2004. 3D-catFISH a system for automated quantitative three-dimensional compartmental analysis of temporal gene transcription activity imaged by fluorescence in situ hybridization. J Neurosci Meth 139 13-24. 

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. 

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. 

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

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