The quantification of ADAMTS4 and 8 expression and selection of reference genes for quantitative real-time PCR analysis in myocardial infarction.
BACKGROUND
ADAMTS4 and ADAMTS8 are proteases involved in ECM proteolysis and antiangiogenesis, but little is known about their expression and function in myocardial infarction (MI). We examined ADAMTS4 and ADAMTS8 expression in a rat MI model by quantitative real-time polymerase chain reaction (qPCR) and enzyme linked immunosorbent assay (ELISA). The expressions of glyseraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB), acidic ribosomal phosphoprotein P0 (ARBP), and ribosomal protein L13A (RPL13A) were examined in order to validate the appropriate housekeeping genes after MI.


METHODS
Male Wistar rats were subjected to MI, and infarcted myocardial tissue was collected at 3, 6, 12, 24h, 3, 7, 14 and 21days after MI. ADAMTS4, ADAMTS8, and the four housekeeping genes were quantified using qPCR and the expression stability of the four housekeeping genes was investigated using GeNorm software. The protein levels of ADAMTS4 were detected using ELISA kits.


RESULTS
The M values of GAPDH, ACTB, ARBP and RPL13A were 0.721, 1.2, 0.812 and 0.812 respectively. GAPDH and ARBP were ranked the most stable genes. ADAMTS4 mRNA increased at 3h after MI, peaked at 6h, then decreased rapidly. ADAMTS8 mRNA increased at 6h, peaked at 24h, remained high at 3d, then decreased gradually. The protein levels of ADAMTS4 were significantly increased at 6h, 12h, 24h and 3d after MI.


CONCLUSIONS
The results suggest that GAPDH and ARBP are two appropriate housekeeping genes for the rat MI model. Both ADAMTS4 and ADAMTS8 mRNA levels and ADAMTS4 protein level increased, but they exhibited different expression profiles.
Department of Cardiology, Shandong Provincial Hospital, Shandong University, 324, Jingwu Road, Jinan 250021, PR China.
Chuanyan
C
Zhao
Yanping
Y
Zha
Xiaoyan
X
Wu
Liming
L
Chen
Jinzheng
J
Shi
Lianqun
L
Cui
D051722:ADAM Proteins / Q000235:genetics*; D000071096:ADAMTS Proteins; D000071121:ADAMTS4 Protein; D000818:Animals; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D008297:Male; D009203:Myocardial Infarction / Q000235:genetics* / Q000473:pathology; D016133:Polymerase Chain Reaction; D011348:Procollagen N-Endopeptidase / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012269:Ribosomal Proteins / Q000235:genetics; D013997:Time Factors
D012333:RNA, Messenger; D012269:Ribosomal Proteins; C054675:ribosomal protein P0; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D051722:ADAM Proteins; D000071096:ADAMTS Proteins; C571365:ADAMTS4 protein, rat; C571366:ADAMTS8 protein, rat; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein