Differential mRNA display

Cirelli C et al. Differences in brain gene expression between sleep and waking as revealed by mRNA differential display and cDNA microarray technology. J Sleep Res 1999 8(Suppl l] 44-52. 

Bhattacharjee A et al. Molecular dissection of dimethylnitrosamine (DMN)-in-duced hepatotoxicity by mRNA differential display. Toxicol Appl Pharmacol 1998 150 186-195. 

The Use of a Concerted RT-PCR mRNA Differential Display Screening Strategy 401... 

Fig. 20.1 mRNA RT-PCR differential display. The figure on the left provides a representative example of the mRNA RT-PCR DD strategy utilized to identify genes whose expression is regulated in concert by both chronic lithium and VPA. The arrows indicate two bands whose levels are markedly increased by both... 

Generally differential display is used to compare levels of gene expression among experimental groups. However, this technique has also been used successfully to reveal mRNA sequence variation between samples. Eor example, differential display demonstrated that a cDNA fragment could be amphfied from samples derived from hippocampi of seizure-resistant but not seizure-prone rats (O Figure 17-2). A smaller... 

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. 

Quantitative RT PCR (qRT PCR) can be used to accurately determine the levels of messages within given preparations of RNA. qRT PCR thermocyclers provide rapid online detection and quantification of mRNA, however, the initial purchase cost and the cost of reagents may be prohibitive for some laboratories. Methods of semiquantitative RT PCR have been used and good descriptions of these techniques are available (Samhrook and Russell, 2001). However, the same cDNA populations should not be used for differential display reactions and verification that a potential differential display band represents a differentially expressed gene. For this reason, independent cDNA samples should be prepared if both the screening and verification methods rely on PCR. qRT PCR, therefore, should be used in conjunction with other methods to verify that a differential display band represents a differentially expressed gene. 

Liang P, Averboukh L, Pardee AB. 1993. Distribution and cloning of eukaryotic mRNAs by means of differential display refinements and optimization. Nucleic Acids Res... 

Simon H-G, Oppenheimer S. 1996. Advanced mRNA differential display isolation of a new differentially regulated myosin heavy chain-encoding gene in amphibian limb regeneration. Gene 172 175. 

Protein expression profiling (protein differential display) using microarrays is considered an important new tool for proteomic discovery. It is similar in concept and approach to the gene expression microarray for mRNA profiling. Sreekumar and Chinnaiyan (2002) describe a general approach for using the microarray to monitor protein expression in cancer and normal tissues. Here are the steps ... 

Yamazaki, M. et al., Molecular cloning and biochemical characterization of a novel anthocyanin 5-0-glucosyltransferase by mRNA differential display for plant forms regarding anthocyanin. J. Biol Chem., 21 A, 7405, 1999. 

Tang, L. Komoroski, B. Tiegler, K. Tibbits, J. Nolan, T. Suizu, R. Neft, R. Kier, L. Strom, S. Li, A.P. A comparison of array hybridization, mRNA differential display and real-time PCR in the evaluation of the effects of Troglita-zone on gene expression in primary human hepatocytes. Toxicologist 2003, 72 (SI), 149. 

Blanchard, R. K., and Cousins, R. J. (1996). Differential display of intestinal mRNAs regulated by dietary zinc. Proc. Natl. Acad. Sci. 93, 6863-6868. 

Fig. 4. Representative segment of a differential display (DD) RT-PCR gel of liver mRNA from fish treated either by injection with growth hormone (GH), triiodothyronine (T3), or given an aquatic exposure to estradiol (E2), chlorpyrifos (CP), or triethylene glycol carrier control (TEG Control). Arrows denote gel bands that were subsequently cloned and sequenced. IK3 represents a gene that appears up-regulated in response to chlorpyrifos (CP) treatment. IK4 represents a gene up-regulated by E2 treatment and is also present in female fish of other treatment groups. IK6 represents a gene that is present in all treatment groups. The sex of the individual fish is denoted (M or F) at the top of each gel lane.

Davis, W., Jr., De Sousa, P.D., and Schultz, R.M. (1996). Transient expression of translation initiation factor elF-4C during the two-cell stage of the preimplantation mouse embryo Identification by mRNA differential display and the role of DNA replication. Dev. Biol. 774 190-201. 

Various methods are available for detecting and quantifying gene-expression levels, including northern blots, differential display, polymerase chain reaction after reverse transcription of RNA, and serial analysis of gene expression. These techniques are used primarily to measure the expression levels of specific genes or to screen for significant differences in mRNA abundance. 

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