Abstract
RNA differential display (DD) RT-PCR is a useful method to identify and clone differentially expressed genes. However, the rate of false positives and redundancy associated with this PCR-based method as well as laborious downstream screening steps constitute major limitations. Here we present DD RT-PCR and reverse northern (RN) protocols allowing rapid and acurate identification of genes upregulated in porcine endothelial cells (EC) in response to TNFalpha. The housekeeping gene beta-actin was used to investigate mispriming and to set up optimal conditions for DD-RT-PCR and RN. In this study DD was performed to compare resting and TNFalpha-activated ECs. Selection of DD-fragments was performed following 30-cycles of PCR using serial dilutions of template cDNA and regulation of 6 out of 17 candidates genes were first confirmed by semi-quantitative RN. Using this protocol, 5 out of 6 DD-fragments were further confirmed to be upregulated by Northern blot, and 3 novel porcine cDNAs were cloned including the pro-apoptotic member of the Bcl-2 family, Noxa. In this study we demonstrate that the combination of DD-RT-PCR and RN, which efficiently reduces the number of false positive candidates derived from mispriming at the screening step, allows a rapid identification of differentially expressed genes.
Highlights
RNA differential display (DD) reverse transcription (RT)-polymerase chain reactions (PCR) is a useful tool to identify differentially expressed genes in various biological systems [1,2,3]
A variety of bands amplified from a single mRNA may be visible on gels, leading to DDfragments which do not reside at the 3’-end of the mRNA. To rule out these possibilities, we present a DD-RT-PCR protocol associated with several criteria for the selection of DD-fragments, aimed to reduce the number of candidate genes selected
We evaluate whether PCR conditions, including number of amplification cycles and template concentration, could influence the detection of a highly expressed housekeeping gene, -actin. [33P] dATPlabeled RT-PCR of porcine -actin was performed with a specific primer set in a high stringency condition
Summary
RNA differential display (DD) RT-PCR is a useful tool to identify differentially expressed genes in various biological systems [1,2,3] This technique has many advantages including the small amounts of RNA samples required, its rapid and easy technical procedure, the possible multiple comparison between samples in one study, the sequence dependent amplification, and its low financial cost, compared to other techniques. We present DD RT-PCR and reverse northern (RN) protocols allowing rapid and acurate identification of genes upregulated in porcine endothelial cells (EC) in response to TNF␣. Conclusion: In this study we demonstrate that the combination of DD-RT-PCR and RN, which efficiently reduces the number of false positive candidates derived from mispriming at the screening step, allows a rapid identification of differentially expressed genes
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