Non-radioisotopic differential display method to directly visualize and amplify differential bands on nylon membrane.

Differential display is a breakthrough in gene isolation methods (1,2). Compared with other gene isolation methods, it is powerful and relatively rapid. The method achieves isolation of differentially expressed genes by using four 3′-end degenerate primers and 20–40 5′-end arbitrary primers to perform RT–PCR. The PCR products are separated on a DNA sequencing gel and the differences between two or more cell lines are visualized and compared on an X-ray film. To isolate the differential bands from other closely spaced bands on a DNA sequencing gel, one must hold the X-ray film in one hand and cut the bands invisible to the eyes from a transparent gel or filter paper using the X-ray film as a reference. A misjudged band excision wastes days of effort and causes frustration in the subsequent Northern hybridization. To avoid unnecessary frustration and health hazardous radioisotopes, we have developed a non-radioisotopic method to display differentially expressed cDNA bands. To illustrate the method, a series of human lung adenocarcinoma cell lines (CL1, CLl-2 and CL1-5) with different invasive capabilities are used. Cells are collected by centrifuging at 300 g for 10 min and total RNA is extracted by using RNAzol B (Biotecx Laboratories, TX). The mRNA differential display method is performed essentially as described by Liang et al. (2) but with some modifications. Five μg of total RNA extracted from cell lines are incubated at 65 C for 15 min before reverse transcription is carried out. 100 pmol of digoxigenin (DIG) conjugated poly-T degenerate primers, e.g., DIG-T12VG, are reverse transcribed in the presence of 0.5 mM dNTP, 10 mM DTT, 0.5 U/μl RNasin (GIBCO-BRL; Gaithersburg, MD), and 200 U of MMLV reverse transcriptase (GIBCO-BRL; Gaithersburg, MD). Reverse transcription is performed at 45 C for 60 min and is stopped by heating to 95 C for 5 min. The sample is then chilled on ice and diluted to 200 μl final volume. To amplify DNA, 2 μl of the 200 μl sample is subjected to a 40-cycle PCR amplification with the annealing temperature set at 38 C. After PCR amplification, samples are separated on a 6% SDS–polyacrylamide gel and transferred onto a nylon membrane using a semi-dry electro-blotting method. Before electro-blotting, the gel with one glass plate still attached is soaked in a transfer buffer (80 mM Tris–HCl, 120 mM borate, 2.5 mM EDTA, pH 8.3) for 30 min. The gel is then attached to a 3 mm filter paper and Figure 1. Non-radioisotopic differential display of three tumor cell lines with different invasive capabilities on a piece of nylon membrane. The primer sets used to generate the cDNA bands are shown on the top of the figure. The arrow heads indicate the differences between the three cell lines.