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Rapid selection of differentially expressed genes in TNF[alpha]-activated endothelial cells
Takaharu Nagasaka1, Gwénola Boulday, Christopher C Fraser
1Institut National de la Recherche Médicale and Institut de Transplantation et de Recherche en Transplantation, Nantes, FR.
Background:
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.
Materials And Methods:
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.
Results:
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.
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.