Selection of endogenous control genes for normalization of gene expression analysis after experimental brain trauma in mice.
Quantitative measurements of gene expression require correction for tissue sample size, RNA quantity, and reverse transcription efficiency. This can be achieved by normalization with control genes. The study was designed to identify candidates not altered after brain trauma. Male C57Bl/6 mice were anesthetized with isoflurane, and a pneumatic brain trauma was induced by controlled cortical impact (CCI) on the right parietal cortex. Brains were removed at 15 min, and 3, 6, 12 and 24 h after CCI and from naive animals (n = 6 each). Absolute copies of six control genes (beta-2-microglobin [B2M], cyclophilin A, beta-actin, hypoxanthine ribosyltransferase [HPRT], porphobilinogen deaminase [PBGD], and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and one example target gene (iNOS) were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR; Lightcycler) in the traumatic focus and contralateral tissue. Control gene expression was stable until 12 h after CCI. At 24 h after CCI expression of B2M, cyclophilin A and HPRT remained stable in the contusion, while expression of beta-actin, GAPDH, and PBGD increased. Due to variations between animals (+/-85%), increases in beta-actin (+64%) and GAPDH (+59%) did not reach the level of significance. In non-contused tissue, expression of all genes dropped 24 h after CCI (range, -17% to -61%). Due to low variations between animals and stable expression after CCI, B2M and cyclophilin A seem to be suitable to serve as single normalizer. Normalization of the example target gene iNOS resulted in varying relative expression extending from onefold (PBDG) to 10-fold (HPRT). The results suggest that the knowledge of the temporal profile of control genes is essential to properly interpret results of mRNA quantification.
Thal
Serge C
SC
Department of Anesthesiology, Johannes Gutenberg University, Mainz, Germany. thal@uni-mainz.de
Wyschkon
Sebastian
S
Pieter
Dana
D
Engelhard
Kristin
K
Werner
Christian
C
D000199:Actins / Q000235:genetics; D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000503:physiopathology; D001923:Brain Chemistry / Q000235:genetics*; D001930:Brain Injuries / Q000235:genetics* / Q000378:metabolism* / Q000503:physiopathology; D021983:Cyclophilins / Q000235:genetics; D004195:Disease Models, Animal; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000235:genetics*; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000235:genetics; D011163:Hydroxymethylbilane Synthase / Q000235:genetics; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors; D015854:Up-Regulation / Q000235:genetics; D001613:beta 2-Microglobulin / Q000235:genetics
D000199:Actins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D001613:beta 2-Microglobulin; D052247:Nitric Oxide Synthase Type II; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+); D007041:Hypoxanthine Phosphoribosyltransferase; D011163:Hydroxymethylbilane Synthase; D021983:Cyclophilins