Isolation of differentially expressed genes in human heart tissues.
We applied RNA arbitrarily primed-PCR (RAP-PCR) to screen the genes differentially expressed between common congenital heart defects (CHD) [atrial septal defect, ventricular septal defect, Tetrology of Fallot (TOF)] and normal human heart samples. Three of these differentially amplified fragments matched cDNA sequences coding for proteins of unknown function in humans: hCALO (human homologue of calossin), NP79 (coding for a nuclear protein of 79KD) and SUN2 (Sad-1 unc-84 domain protein 2). The other four fragments were from known human genes: apolipoprotein J, titin, dystrophin and protein kinase C-delta. Northern blot analysis confirmed that all of these genes are expressed in the human heart. The results of RAP-PCR were reconfirmed by quantitative RT-PCR in TOF and control heart samples. Both techniques showed the levels of expression of hCALO, NP79 and SUN2 to be comparable in TOF and control samples and the level of expression of dystrophin and titin, both coding for cytoskeletal proteins, to be significantly upregulated in TOF samples. In summary, we have shown that the RAP-PCR technique is useful in the identification of differentially expressed gene from biopsy samples of human CHD tissues. In this manner, we have identified three novel genes implicated in the normal function of the human heart and two known genes upregulated in TOF samples.
Sun
Guifeng
G
Department of Medical Genetics, Institute of Basic Medicine, China Medical University, Shenyang, PR China. guifengs@uci.edu
Yuen Chan
Siu
S
Yuan
Yihua
Y
Wang Chan
Kin
K
Qiu
Guangrong
G
Sun
Kailai
K
Ping Leung
Maurice
M
D002148:Calmodulin-Binding Proteins / Q000235:genetics; D002648:Child; D002675:Child, Preschool; D003001:Cloning, Molecular; D064211:Connectin; D003327:Coronary Disease / Q000235:genetics* / Q000503:physiopathology*; D018076:DNA, Complementary / Q000032:analysis; D029721:Drosophila Proteins; D016189:Dystrophin / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006321:Heart / Q000254:growth & development / Q000503:physiopathology*; D006344:Heart Septal Defects, Atrial / Q000235:genetics; D006345:Heart Septal Defects, Ventricular / Q000235:genetics; D006801:Humans; D007223:Infant; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000032:analysis; D015203:Reproducibility of Results; D013771:Tetralogy of Fallot / Q000235:genetics; D044767:Ubiquitin-Protein Ligases
D002148:Calmodulin-Binding Proteins; D064211:Connectin; D018076:DNA, Complementary; D029721:Drosophila Proteins; D016189:Dystrophin; D009124:Muscle Proteins; D009687:Nuclear Proteins; C577285:TTN protein, human; D012313:RNA; D044767:Ubiquitin-Protein Ligases; C115917:poe protein, Drosophila; D011494:Protein Kinases