title: Effective Alu repeat based RT-Qpcr normalization in cancer cell perturbation experiments.
abstract: BACKGROUND
  Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in gene expression levels, even of assumed reference genes. In this study, we evaluated the expression levels of 11 commonly used reference genes as internal controls for normalization of 19 experiments that include neuroblastoma, T-ALL, melanoma, breast cancer, non small cell lung cancer (NSCL), acute myeloid leukemia (AML), prostate cancer, colorectal cancer, and cervical cancer cell lines subjected to various perturbations.
  
  
  RESULTS
  The geNorm algorithm in the software package qbase+ was used to rank the candidate reference genes according to their expression stability. We observed that the stability of most of the candidate reference genes varies greatly in perturbation experiments. Expressed Alu repeats show relatively stable expression regardless of experimental condition. These Alu repeats are ranked among the best reference assays in all perturbation experiments and display acceptable average expression stability values (M<0.5).
  
  
  CONCLUSIONS
  We propose the use of Alu repeats as a reference assay when performing cancer cell perturbation experiments.
authors: Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.
authors: Ali
authors: A
authors: Rihani
authors: Tom
authors: T
authors: Van Maerken
authors: Filip
authors: F
authors: Pattyn
authors: Gert
authors: G
authors: Van Peer
authors: Anneleen
authors: A
authors: Beckers
authors: Sara
authors: S
authors: De Brouwer
authors: Candy
authors: C
authors: Kumps
authors: Evelien
authors: E
authors: Mets
authors: Joni
authors: J
authors: Van der Meulen
authors: Pieter
authors: P
authors: Rondou
authors: Carina
authors: C
authors: Leonelli
authors: Pieter
authors: P
authors: Mestdagh
authors: Frank
authors: F
authors: Speleman
authors: Jo
authors: J
authors: Vandesompele
mesh_terms: D020087:Alu Elements; D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D017124:Conserved Sequence; D020869:Gene Expression Profiling / Q000592:standards*; D055785:Gene Knockdown Techniques; D006801:Humans; D035683:MicroRNAs / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D012097:Repressor Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D059467:Transcriptome / Q000187:drug effects; D014212:Tretinoin / Q000494:pharmacology; D054358:Withanolides / Q000494:pharmacology
chemical_list: D020533:Angiogenesis Inhibitors; D002352:Carrier Proteins; D035683:MicroRNAs; C516714:NVP-TAE684; C502542:PHF6 protein, human; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D054358:Withanolides; D014212:Tretinoin; C009684:withaferin A
references: Methods. 2010 Apr;50(4):227-30
references: 19969088
references: Anal Biochem. 2005 Sep 1;344(1):141-3
references: 16054107
references: BMC Bioinformatics. 2009 Feb 19;10:62
references: 19228411
references: Cancer Res. 1999 Jun 15;59(12):2759-65
references: 10383126
references: Anal Biochem. 2002 Oct 15;309(2):293-300
references: 12413463
references: Cancer Lett. 2010 Nov 28;297(2):220-5
references: 20576349
references: Mol Cancer Ther. 2011 Jun;10(6):983-93
references: 21460101
references: Genomics. 2004 Aug;84(2):441-8
references: 15234007
references: Biochemistry. 1980 Jul 22;19(15):3452-60
references: 6157399
references: Anal Biochem. 2001 Jan 1;288(1):99-102
references: 11141312
references: Nature. 2005 Feb 17;433(7027):769-73
references: 15685193
references: J Exp Clin Cancer Res. 2010 May 11;29:45
references: 20459794
references: Circ Res. 2000 Sep 1;87(5):355-62
references: 10969032
references: Nucleic Acids Res. 2009 Jan;37(Database issue):D942-5
references: 18948285
references: Clin Chem. 2009 Apr;55(4):611-22
references: 19246619
references: Genome Biol. 2002 Jun 18;3(7):RESEARCH0034
references: 12184808
references: Nat Genet. 2011 Jun 05;43(7):673-8
references: 21642990
references: BMC Genomics. 2008 Jul 11;9:328
references: 18620571
references: Genome Biol. 2007;8(2):R19
references: 17291332
references: Cell Death Differ. 2009 Dec;16(12):1563-72
references: 19779493
references: Biochem Soc Trans. 2004 Dec;32(Pt 6):952-6
references: 15506933
references: J Pediatr Surg. 2008 Jul;43(7):1284-94
references: 18639684
references: Chem Biol. 2007 Jun;14(6):623-34
references: 17584610
references: Transl Oncol. 2011 Aug;4(4):258-65
references: 21804922
references: Proc Natl Acad Sci U S A. 2007 Jan 2;104(1):270-5
references: 17185414
references: BMC Dev Biol. 2005 Dec 03;5:27
references: 16324220