title: Simple regression for correcting ΔCt bias in RT-qPCR low-density array data normalization.
abstract: BACKGROUND
  Reverse transcription quantitative PCR (RT-qPCR) is considered the gold standard for quantifying relative gene expression. Normalization of RT-qPCR data is commonly achieved by subtracting the Ct values of the internal reference genes from the Ct values of the target genes to obtain ΔCt. ΔCt values are then used to derive ΔΔCt when compared to a control group or to conduct further statistical analysis.
  
  
  RESULTS
  We examined two rheumatoid arthritis RT-qPCR low density array datasets and found that this normalization method introduces substantial bias due to differences in PCR amplification efficiency among genes. This bias results in undesirable correlations between target genes and reference genes, which affect the estimation of fold changes and the tests for differentially expressed genes. Similar biases were also found in multiple public mRNA and miRNA RT-qPCR array datasets we analysed. We propose to regress the Ct values of the target genes onto those of the reference genes to obtain regression coefficients, which are then used to adjust the reference gene Ct values before calculating ΔCt.
  
  
  CONCLUSIONS
  The per-gene regression method effectively removes the ΔCt bias. This method can be applied to both low density RT-qPCR arrays and individual RT-qPCR assays.
authors.affiliation: Department of Biostatistics, School of Public Health, University of Alabama at Birmingham, Birmingham, AL, 35294, USA. xcui@uab.edu.
authors.forename: Xiangqin
authors.initials: X
authors.lastname: Cui
authors.affiliation: Department of Biostatistics, School of Public Health, University of Alabama at Birmingham, Birmingham, AL, 35294, USA. yus@uab.edu.
authors.forename: Shaohua
authors.initials: S
authors.lastname: Yu
authors.affiliation: Department of Biostatistics, School of Public Health, University of Alabama at Birmingham, Birmingham, AL, 35294, USA. ATamhane@uab.edu.
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Tamhane
authors.affiliation: Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA. zcausey@uab.edu.
authors.forename: Zenoria L
authors.initials: ZL
authors.lastname: Causey
authors.affiliation: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA. asteg@uab.edu.
authors.forename: Adam
authors.initials: A
authors.lastname: Steg
authors.affiliation: Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA. mdanila@uab.edu.
authors.forename: Maria I
authors.initials: MI
authors.lastname: Danila
authors.affiliation: Department of Biostatistics, School of Public Health, University of Alabama at Birmingham, Birmingham, AL, 35294, USA. rjriv@uab.edu.|Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA. rjriv@uab.edu.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Reynolds
authors.affiliation: Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA. jinw@uab.edu.
authors.forename: Jinyi
authors.initials: J
authors.lastname: Wang
authors.affiliation: Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA. kwanzeck@uab.edu.
authors.forename: Keith C
authors.initials: KC
authors.lastname: Wanzeck
authors.affiliation: Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA. tangqi@uab.edu.|Department of Rheumatology and Immunology, Second Xiangya Hospital of Central South University, Changsha, P. R. China. tangqi@uab.edu.
authors.forename: Qi
authors.initials: Q
authors.lastname: Tang
authors.affiliation: Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA. sledbetter@uab.edu.
authors.forename: Stephanie S
authors.initials: SS
authors.lastname: Ledbetter
authors.affiliation: Department of Biostatistics, School of Public Health, University of Alabama at Birmingham, Birmingham, AL, 35294, USA. samndave@uab.edu.
authors.forename: David T
authors.initials: DT
authors.lastname: Redden
authors.affiliation: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA. martinjohnsonmrj@gmail.com.
authors.forename: Martin R
authors.initials: MR
authors.lastname: Johnson
authors.affiliation: Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA. lbridges@uab.edu.
authors.forename: S Louis
authors.initials: SL
authors.lastname: Bridges
mesh_terms: D001172:Arthritis, Rheumatoid / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000378:metabolism; D012015:Reference Standards; D012044:Regression Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger