Fluorescent detection of differentially expressed cDNA using SYBR gold nucleic acid gel stain.

We describe herein a modified differential gene display (DGD) technique that can be rapidly and simply performed and that eliminates the need for radioactivity by fluorescent visualization of complementary deoxyribonucleic acid (cDNA) bands with SYBR gold nucleic acid gel stain. To streamline the DGD procedure, a number of modifications were employed. Ribonucleic acid isolated from differentially treated populations of human trabecular bone-derived mesenchymal progenitor cells was reverse-transcribed into cDNA using oligo-dT primer, and subsequent amplification of differentially expressed cDNAs was done using arbitrary 25-mer primers and oligo-dT9 30-mer primers. Moderate-sized nondenaturing 6% polyacrylamide gels (30 x 20 cm) of 1.5-mm thickness were used for easier handling and increased sample loading capacity. Gels were subjected to electrophoresis overnight, stained with SYBR gold, and visualized and photographed using a commercially available gel imager. DNA bands ranging in size from 100 to 400 bp were visualized directly on an ultraviolet transilluminator, excised from the gel, and reamplified. The cDNA amplicons were subcloned, sequenced, and gene sequences were identified by a Basic Local Alignment Search Tool of genomic databases. Overall, this rapid and functional method proved quite effective for identification of novel genes that may be of interest in studies of cartilage and bone differentiation.