Fluorescent detection of differentially expressed cDNA using SYBR gold nucleic acid gel stain |
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Authors: | Keith G Danielson Shirisha Kanthala Richard Tuli Rocky S Tuan |
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Institution: | (1) Department of Orthopaedic Surgery, Thomas Jefferson University, Room 501 Curtis Bldg., 1015 Walnut St., 19107 Philadelphia, PA;(2) Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, 20892-5755 Bethesda, MD |
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Abstract: | 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 × 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. |
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Keywords: | mRNA differential display PCR polyacrylamide gel SYBR Gold staining |
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