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The specificity of the SOLIDscript™ cDNA Synthesis Kit for solid phase RT-PCR assays was evaluated by looking at the tissue-specific expression of the dopamine receptor D2 gene and the tyrosine hydroxylase (TH) gene. Both of these genes have been previously documented to be exclusively expressed in neural tissue^1,2. Immobilized cDNA libraries were made directly from mouse brain and liver, and then amplified by PCR. In Figure 1, absolute tissue-specificity is confirmed. Duplicate RT-PCR reactions for D2 (lanes 1-2) and TH (lanes 3-4) from liver tissue showed no amplicon, whereas duplicates from brain tissue (lanes 5-8) produced amplicons of the expected sizes. The lack of any spurious bands in the samples from liver is indicative of the specificity of the RT-PCR reactions performed using SOLIDscript™ .

Figure 1: Specificity of Solid Phase RT-PCR. Immobilized cDNA libraries were constructed from the liver (lanes 1-4) and brain (lanes 5-8) of the same mouse using the SOLIDscript™ Solid Phase cDNA Synthesis Kit. PCR amplification (35 cycles) was performed using the Taq-FORCE™ Amplification System with either D2 (lanes 1,2,5,6) or TH (lanes 3,4,7,8) mouse primer pairs. PCR products were resolved on a 1.2% agarose gel (1X TAE) and visualized by ethidium bromide staining.

RT-PCR is viewed as a simple and robust method for comparative analysis of gene expression allowing researchers to readily study both differential levels of mRNA expression in different tissues, and varying levels of expression between different genes in the same tissue. PureBiotech LLC's SOLIDscript™ Solid Phase cDNA Synthesis Kit takes RT-PCR to a new level in simplicity while maintaining equal to or greater sensitivity over standard commercially available methods. The kit provides a streamlined approach that allows you to start with tissue, cells, or total RNA and end up with an immobilized cDNA library in 90 minutes. A small fraction of the immobilized library is then directly mixed with PCR reagents and results can be visualized three to three and one half hours later on an ethidium bromide-stained gel. The procedure allows you to go from cells to RT-PCR in six hours with minimal manipulations.

The SOLIDscript™ procedure involves the following steps: 1) binding of biotinylated oligo(dT)₂₅ to streptavidin MPG® ; 2) lysing the tissue or cells; 3) hybridizing the MPG®-immobilized oligo(dT) to the mRNA directly in the lysate; and 4) after a quick series of washes, incubation with AMV-RT at 42-50°C for one hour. The rapid magnetic separation steps and the elimination of the mRNA elution step make this protocol fast and easy.

The immobilized cDNA libraries generated by the SOLIDscript™ kit can be used in a variety of applications including RACE, general amplification of all of the cDNA, cDNA cloning and, most commonly, PCR of specific target genes.

¹ Monsma, F.J.Jr., McVittie, L.D., Gerfen, C.R., Mahan, L.C. and Sibley, D.R. (1989) Nature 342: 926-927.
² Kumer, S.C. and Vrana, K.E. (1996) Journal of Neurochemistry 67: 443-462.

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