A protocol for differential display of mRNA expression using either fluorescent or radioactive labeling

Abstract

Since its invention in the early 1990s, differential display (DD) has become one of the most commonly used techniques for identifying differentially expressed genes at the mRNA level. Unlike other genomic approaches, such as DNA microarrays, DD systematically detects changes in mRNA profiles among multiple samples being compared without the need of any prior knowledge of genomic information of the living organism being studied. Here, we present an optimized DD protocol with a fluorescent digital readout as well as traditional radioactive labeling. The resulting streamlined fluorescent DD process offers an unprecedented accuracy, sensitivity and throughput in comprehensive and quantitative analysis of eukaryotic gene expression. Results usually can be obtained within days using a limited number of primer combinations, but a comprehensive DD screen may take weeks or months to accomplish, depending on gene coverage required and the number of differentially expressed genes present within a biological system being compared.

Cold stress alters transcription in meiotic anthers of cold tolerant chickpea (Cicer arietinum L.)

“…Amplifications were carried out in a Perkin Elmer Thermal Cycler (Gene Amp PCR System 9700) using 1 cycle of 4 min at 94°C followed by 39 cycles of 15 sec at 94°C (denaturation), 2 min at 40°C (primer annealing) and a 30 sec extension at 72°C followed by a cycle of 72°C for 8 min using the procedure as outlined by Liang et al . [ 46 ] with slight modifications. While Liang et al .…”

Identification and characterization of REC66, a Ty1-copia-like retrotransposon in the genome of red flower of Mirabilis jalapa L.

“…FDD-PCR was carried out for the comparison of mRNA expression profiles between the flower and leaves of the red M. jalapa. Combinations of one-base anchored oligo (dT) primers and arbitrary 13-mers with fluorescent labels from the RNA spectra kits (GenHunter Corp.) were used for the FDD screening [12]. cDNAs that were amplified from the 3' terminal of the mRNAs were separated on a 6% denaturing polyacrylamide gel, and visualized via fluorescent imaging on a Typhoon 9410 imager (GE biosciences).…”

S-phase-coupled apoptosis in tumor suppression

“…Killin was discovered in an attempt to systematically identify p53 target genes through high-throughput fluorescent differential display (FDD) screening using two tetracycline inducible cell lines [107][108][109][110][111], the DLD-1 colon cancer cell line [112,113] and the p53-null human lung carcinoma cell line H1299 [114]. Both cell lines contained tetracycline-regulated expression of the wildtype p53 tumor suppressor gene and underwent apoptosis within 24-48 h after removal of tetracycline.…”