title: Toward enhanced MIQE compliance: reference residual normalization of qPCR gene expression data.
abstract: Normalization of fluorescence-based quantitative real-time PCR (qPCR) data varies across quantitative gene expression studies, despite its integral role in accurate data quantification and interpretation. Identification of suitable reference genes plays an essential role in accurate qPCR normalization, as it ensures that uncorrected gene expression data reflect normalized data. The reference residual normalization (RRN) method presented here is a modified approach to conventional 2(-ΔΔCt)qPCR normalization that increases mathematical transparency and incorporates statistical assessment of reference gene stability. RRN improves mathematical transparency through the use of sample-specific reference residuals (RR i ) that are generated from the mean Ct of one or more reference gene(s) that are unaffected by treatment. To determine stability of putative reference genes, RRN uses ANOVA to assess the effect of treatment on expression and subsequent equivalence-threshold testing to establish the minimum permitted resolution. Step-by-step instructions and comprehensive examples that demonstrate the influence of reference gene stability on target gene normalization and interpretation are provided. Through mathematical transparency and statistical rigor, RRN promotes compliance with Minimum Information for Quantitative Experiments and, in so doing, provides increased confidence in qPCR data analysis and interpretation.
authors: Ecotoxicology Program, Environmental Conservation Division, and.
authors: Richard C
authors: RC
authors: Edmunds
authors: Washington State University Puyallup Research and Extension Center, Puyallup, Washington 98371, USA; and.
authors: Jenifer K
authors: JK
authors: McIntyre
authors: Environmental Physiology Program, Resource Enhancement and Utilization Technologies Division, Northwest Fisheries Science Center, National Oceanic and Atmospheric Administration, Seattle, Washington 98112, USA; ; Center for Reproductive Biology, Washington State University, Pullman, Washington 99164, USA.
authors: J Adam
authors: JA
authors: Luckenbach
authors: Ecotoxicology Program, Environmental Conservation Division, and.
authors: David H
authors: DH
authors: Baldwin
authors: Ecotoxicology Program, Environmental Conservation Division, and.
authors: John P
authors: JP
authors: Incardona
mesh_terms: D000704:Analysis of Variance; D003627:Data Interpretation, Statistical; D015870:Gene Expression; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards* / Q000706:statistics & numerical data; D012015:Reference Standards
keywords: ANOVA; normalization; reference genes; threshold cycle; treatment effect