Reference genes identified in SH-SY5Y cells using custom-made gene arrays with validation by quantitative polymerase chain reaction.
Transcriptomic methods are widely used as an initial approach to gain a mechanistic insight into physiological and pathological processes. Because differences in gene regulation to be assessed by RNA screening methods (e.g., SAGE, Affymetrix GeneChips) can be very subtle, these techniques require stable reference genes for accurate normalization. It is widely known that housekeeping genes, which are routinely used for normalization, can vary significantly depending on the tissue, and experimental test. In this study, we aimed at identifying stable reference genes for a fibrillar Abeta(42) peptide-treated, human tau-expressing SH-SY5Y neuroblastoma cell line derived to model aspects of Alzheimer's disease in tissue culture. We selected genes exhibiting potential normalization characteristics from public databases to create a custom-made microarray allowing the identification of reference genes for low, intermediate, and abundant mRNAs. A subset of these candidates was subjected to quantitative real-time polymerase chain reaction and was analyzed with geNorm software. By doing so, we were able to identify GAPD, M-RIP, and POLR2F as stable and usable reference genes irrespective of differentiation status and Abeta(42) treatment.
Hoerndli
Frédéric J
FJ
Division of Psychiatry Research, University of Zürich, August Forel Strasse 1, 8008 Zürich, Switzerland.
Toigo
Marco
M
Schild
Andreas
A
Götz
Jürgen
J
Day
Philip J
PJ
D000544:Alzheimer Disease / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000032:analysis* / Q000378:metabolism; D029721:Drosophila Proteins / Q000032:analysis* / Q000378:metabolism; D020869:Gene Expression Profiling; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis / Q000378:metabolism; D006801:Humans; D009447:Neuroblastoma / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010749:Phosphoprotein Phosphatases / Q000032:analysis* / Q000378:metabolism; D012313:RNA / Q000032:analysis / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D016229:Amyloid beta-Peptides; D029721:Drosophila Proteins; D012333:RNA, Messenger; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010749:Phosphoprotein Phosphatases; C079898:tws protein, Drosophila