Validation of two reference genes for mRNA level studies of murine disease models in neurobiology.
Reverse transcription of extracted cellular RNA combined with real-time PCR is now an established method for sensitive detection and quantification of specific mRNA level changes in experimental models of neurological diseases. To neutralize the impact of experimental error and make quantification more precise, normalization of test gene data using data from a constantly expressed gene, a reference gene that is tested along with the test gene, is required. There is no single gene constantly expressed under all experimental conditions. For a given set of conditions or a given disease model, identification of an unaffected reference gene is necessary. In this report, we present our findings from evaluation and validation of the genes encoding hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1) and glyceraldehyde phosphate dehydrogenase (GAPDH) as individual reference genes in mRNA level studies involving four murine neurological disease models. We find both genes are suitable as a reference gene with these four models, provided quantification of subtle changes are avoided. We furthermore demonstrate that above a certain threshold of test mRNA level changes and given high quality RNA processing, normalization to total RNA alone provides for equally reliable quantitative mRNA level results.
Meldgaard
Michael
M
Medical Biotechnology Center, University of Southern Denmark, Winsløwparken 25, DK-5000 Odense, Denmark. mmeldgaard@health.sdu.dk
Fenger
Christina
C
Lambertsen
Kate L
KL
Pedersen
Mads D
MD
Ladeby
Rune
R
Finsen
Bente
B
D000818:Animals; D001369:Axons / Q000502:physiology; D003337:Corpus Callosum; D003627:Data Interpretation, Statistical; D003714:Denervation; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000235:genetics / Q000473:pathology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006624:Hippocampus / Q000502:physiology; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D020244:Infarction, Middle Cerebral Artery / Q000235:genetics / Q000473:pathology; D008244:Lysophosphatidylcholines / Q000008:administration & dosage / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008845:Microinjections; D009422:Nervous System Diseases / Q000235:genetics* / Q000473:pathology; D019580:Perforant Pathway / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013116:Spinal Cord / Q000473:pathology; D014855:Wallerian Degeneration / Q000235:genetics / Q000503:physiopathology
D008244:Lysophosphatidylcholines; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007041:Hypoxanthine Phosphoribosyltransferase