Rapid selection of differentially expressed genes in TNF[alpha]-activated endothelial cells.
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.


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.


CONCLUSIONS
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.
Nagasaka
Takaharu
T
Institut National de la Recherche Médicale and Institut de Transplantation et de Recherche en Transplantation, Nantes, FR.
Boulday
Gwénola
G
Fraser
Christopher C
CC
Coupel
Stéphanie
S
Coulon
Flora
F
Tesson
Laurent
L
Heslan
Jean-Marie
JM
Soulillou
Jean-Paul
JP
Charreau
Béatrice
B
D000818:Animals; D001011:Aorta / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D004730:Endothelium, Vascular / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D008969:Molecular Sequence Data; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013552:Swine; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015854:Up-Regulation
D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha