Differential display PCR

Wang JF. Differential display PCR reveals increased expression of 2, 3 -cyclic nucleotide 3 -phosphodiesterase by lithium. FEBS Lett 1996 386 225-229. 

Wang JF, Young LT. Differential display PCR reveals novel targets for the mood-stabilizing dmg valproate including the molecular chaperone GRP78. Mol Pharmacol 1999 55 521-527. 

O Rourke JF, Pugh CW, Bartlett SM, Ratcblfe PJ. Idenfication of hypoxically inducible mRNAs in HeLa cells using differential-display PCR. Eur J Biochem 1996 241 403 10. 

Fig. 19.1 Differential displays comparing RNAs from saline (S)-, imipramine (I)- or fluoxetine (F)-treated rats. Total RNA was extracted from hypothalami of animals treated with the different drugs for two months. Autoradiograms of amplified -[35S]-dATP-labeled PCR (polymerase chain reaction) products after electrophoresis in 6% polyacrylamide gels are shown for two different primer combinations that identified one upregulated (arrowhead) and one downregulated (arrow) fragment in the groups treated with antidepressants (from [4] with permission).

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... 

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. 

Joshi, L., St. Leger, R. J., and Roberts, D. W. (1997). Isolation of a cDNA encoding a novel subtilisin-like protease (PrlB) from the entomopathogenic fungus, Metarhizium anisopliae using differential display-RT-PCR. Gene, 197, 1-8. 

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. 

Differential display RT-PCR — another open method to analyze differentially expressed genes... 

---