Regulation of hypoxanthine phosphoribosyltransferase, glyceraldehyde-3-phosphate dehydrogenase and beta-actin mRNA expression in porcine immune cells and tissues.
Various "housekeeping" genes are often used as endogenous controls in gene expression experiments. We have cloned from swine, three genes commonly used as endogenous controls in other species and have characterized their relative levels of expression in various porcine tissues and their response to various cell activators. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin were readily detected by northern hybridization in various tissues and in alveolar macrophages. The expression of hypoxanthine phosphoribosyltransferase (HPRT) was detected only by northern hybridization of poly-A+ enriched RNA and by reverse transcriptase-polymerase chain reaction (RT-PCR), making it more suitable for highly sensitive detection methods. Expression of GAPDH varied less among tissues than did beta-actin, making it more useful control for comparisons of gene expression between tissues with northern hybridizations. Various treatments of cultured alveolar macrophages differentially affected levels of beta-actin and GAPDH, while HPRT expression was unchanged in alveolar macrophages or spleen cells similarly treated. Therefore, while HPRT can be used as the endogenous control with sensitive detection methods such as RT-PCR, less sensitive detection methods require a more abundant gene such as GAPDH.
Foss
D L
DL
Department of Veterinary PathoBiology, University of Minnesota, St. Paul 55108, USA.
Baarsch
M J
MJ
Murtaugh
M P
MP
D000199:Actins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern / Q000662:veterinary; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003208:Concanavalin A / Q000494:pharmacology; D003720:Densitometry / Q000662:veterinary; D005786:Gene Expression Regulation; D015971:Gene Expression Regulation, Enzymologic; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D008070:Lipopolysaccharides / Q000494:pharmacology; D016676:Macrophages, Alveolar / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012333:RNA, Messenger / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013154:Spleen / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D013552:Swine / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D000199:Actins; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D003208:Concanavalin A; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007041:Hypoxanthine Phosphoribosyltransferase