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• From cells to first strand in a single tube in 2 hours
• Complete system, for immobilized cDNA library construction including mRNA purification step
• Each immobilized cDNA library yields 20 robust PCR reactions
• Ready for direct use in PCR, subtractive hybridization, RACE, cDNA amplification, aRNA amplification, and cDNA cloning

mRNA Purification and cDNA Synthesis in a Single Tube in 2 Hours

Introducing SOLIDscript™ Solid Phase cDNA Synthesis Kit⁽¹⁾, allowing you to go from cells, animal or plant tissue, or total RNA directly to an immobilized cDNA library in 2 hours or less. This 2 hour system enables you to complete your PCR² reactions and gel analysis in the same day, with a minimum of 6 hours from cells to results. The immobilized cDNA library is ready for direct use in PCR, subtractive hybridization, RACE, cDNA amplification, aRNA amplification, and cDNA cloning. The kit comes complete with both mRNA purification and first strand synthesis components and enough reagents to complete 25 reactions. Each kit reaction makes an immobilized cDNA library large enough for 20 robust PCR reactions. Each reaction takes anywhere from 90 minutes to 2-1/2 hours depending on the number of samples being processed, and is completed in a single tube with minimal sample loss.

FIGURE 1: Schematic of SOLIDscript™ Solid Phase cDNA Synthesis Kit protocol. Both the mRNA purification and cDNA synthesis steps are carried out on a magnetic solid support.

The first steps involve immobilization of mRNA on streptavidin-coated magnetic particles (MPG®) bound with oligo (dT)₂₅. The mRNA-MPG® particles are used without further modification in a first strand synthesis reaction catalyzed by AMV reverse transcriptase and primed off of the MPG®-bound oligo (dT)₂₅. The result is an immobilized, single-stranded, cDNA library from a streamlined procedure schematically summarized in Figure 1. The entire protocol is accomplished in a single tube, thus eliminating potential loss of template and increasing the likelihood of detecting poorly expressed genes in the generated cDNA library. Other benefits include rapid manipulations with no phenol extractions, ethanol precipitations, or centrifugations. The fact that the cDNA library is immobilized on magnetic particles allows for an easy one-step removal of the template after a PCR reaction.

Solid Phase-Based versus Solution-Based RT-PCR

To prove the efficacy of this kit for RT-PCR applications, we compared detection of the expression of the mouse IL-1ß gene using both our solid phase-based protocol and the standard solution-based protocol. The starting material for this experiment was mouse liver. The mRNA was immobilized on oligo (dT)₂₅-MPG® directly out of a tissue lysate. The material was then split equally, with half remaining on the particles for the solid phase RT-PCR, and half eluted from the particles for use in solution-based RT-PCR. Both the RT and PCR reactions were performed with equivalent amounts of starting material. The PCR products were resolved on a 1.2% agarose gel in 1X TAE, and visualized by ethidium bromide staining. The results shown in Figure 2 demonstrate a slightly higher band intensity for solid phase versus solution-based, as well as a reduction in primer dimer.

FIGURE 2:

Comparison of Solid Phase - Versus Solution Based RT-PCR. cDNA libraries forma a single mouse liver were constructed on a sagnetic solid support using SOLIDscript™ or in solution from isolated mRNA. Equal amounts of cDNA were analyzed for IL-1ß expression by PCR. PCR products were resolved on a 1.2% agarose gel (1X TAE) and visualized by EtBr staining.

Differential Expression Analysis using Solid Phase RT-PCR

The SOLIDscript™ Solid Phase cDNA Synthesis Kit was then used to look at differential expression of three genes in four different mouse tissues. Immobilized cDNA libraries were generated directly from heart, kidney, liver, and lung using the kit protocol, and then each library was used directly in PCR reactions using either ß-actin, IL-1ß, or p53 primer pairs (Figure 3). The results were comparable to results obtained in an analogous assay using solution-phase RT-PCR (data not shown), but with all the benefits of performing the assay on a solid phase. These benefits include minimal sample loss, greater sensitivity, ease of use, and rapid removal of PCR template after amplification.

FIGURE 3:

Differential Expression Analysis by Solid Phase RT-PCR

Immobilized cDNA libraries were constructed from the liver, lung, heart, and kidney of a single mouse using the SOLIDscript™ Solid Phase cDNA Synthesis Kit. Differential gene expression levels of ß-actin, IL-1ß and p53 in the cDNA libraries were analyzed by PCR using our Taq-FORCE™ Amplification System. PCR produts were resolved on a 1.2% agarose gel (1X TAE) and visualized by EtBr staining.

Stable Immobilized cDNA Libraries for Multiple Applications

In addition to the RT-PCR application highlighted in this article, there are many other applications that can benefit from, or are facilitated by, the use of an immobilized cDNA library. For example, immobilized libraries can be used for subtractive hybridization, RACE, cDNA cloning, other methods of cDNA amplification, and aRNA amplification. The kit provides enough reagents for 25 first strand synthesis reactions. The resultant immobilized cDNA libraries are stable under proper storage conditions for a minimum of 3 months, and each library can be used for 20 PCR reactions.

Next Article: pGATA Positive Selection Vector