Analysis of qPCR reference gene stability determination methods and a practical approach for efficiency calculation on a turbot (Scophthalmus maximus) gonad dataset

Abstract

BackgroundGene expression analysis by reverse transcription quantitative PCR (qPCR) is the most widely used method for analyzing the expression of a moderate number of genes and also for the validation of microarray results. Several issues are crucial for a successful qPCR study, particularly the selection of internal reference genes for normalization and efficiency determination. There is no agreement on which method is the best to detect the most stable genes neither on how to perform efficiency determination. In this study we offer a comprehensive evaluation of the characteristics of reference gene selection methods and how to decide which one is more reliable when they show discordant outcomes. Also, we analyze the current efficiency calculation controversy. Our dataset is composed by gonad samples of turbot at different development times reared at different temperatures. Turbot (Scophthalmus maximus) is a relevant marine aquaculture European species with increasing production in the incoming years. Since females largely outgrow males, identification of genes related to sex determination, gonad development and reproductive behavior, and analysis of their expression profiles are of primary importance for turbot industry.ResultsWe analyzed gene stability of six reference genes: RPS4, RPL17, GAPDH, ACTB, UBQ and B2M using the comparative delta-CT method, Bestkeeper, NormFinder and GeNorm approaches in gonad samples of turbot. Supported by descriptive statistics, we found NormFinder to be the best method, while on the other side, GeNorm results proved to be unreliable. According to our analysis, UBQ and RPS4 were the most stable genes, while B2M was the least stable gene. We also analyzed the efficiency calculation softwares LinRegPCR, LREanalyzer, DART and PCR-Miner and we recommend LinRegPCR for research purposes since it does not systematically overestimate efficiency.ConclusionOur results indicate that NormFinder and LinRegPCR are the best approaches for reference gene selection and efficiency determination, respectively. We also recommend the use of UBQ and RPS4 for normalization of gonad development samples in turbot.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-648) contains supplementary material, which is available to authorized users.

An improved method for RNA extraction from common bean seeds and validation of reference genes for qPCR

“…As described by Peirson et al (2003), several analytical procedures could result in a cumulative error that might lead to efficiency overestimation. To overcome these limitations, mathematical models have been published describing the kinetics of the qPCR reaction and trying to estimate qPCR efficiency from a single reaction (Robledo et al 2014). The methodology implemented by the LinRegPCR software used in the present study resulted in values of qPCR efficiency ranging from 55 to 94%, which are reduced values compared to the standard curve.…”

“…The absolute Cq values individually obtained for each one of the five reference genes are graphically represented on a box plot graphic in Figure 1 where the median raw Cqs are represented by lines. There is a premise that reference genes cannot be regulated by the experimental conditions of the sample set (Robledo et al 2014). The geNorm and BestKeeper algorithms are based on the assumption that none of the analyzed genes are co-regulated (Matz et al 2013).…”

“…For all samples analyzed together, geNorm ranked T197 and β-tubulin as the most stable genes (M = 1.4) with the combined value below the recommended cutoff of 1.5 (Robledo et al 2014). NormFinder considered the same two genes most stable, with a combined stability value of 0.468.…”

Docosahexaenoic Acid-Rich Fish Oil Supplementation Improves Body Composition without Influence of the PPARγ Pro12Ala Polymorphism in Patients with Type 2 Diabetes: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial

“…GAPDH was used as housekeeping gene (forward: 5′-TGGTATCGTGGAAGGACTCATG-3′; reverse: 5′-GCTTCACCACCTT-CTT-GATGTC-3′). LinRegPCR software was applied to determine PCR efficiency [36]. The fold changes of gene expression were computed using the Pfaffl method [37].…”

miPrimer: an empirical-based qPCR primer design method for small noncoding microRNA

“…First, a miRNA serial titration assay was performed using synthetic oligonucleotide hsa-miR-9-5p as a template, a forward primer hsa-miR-9-5p-F and a universal reverse primer (pair of primers designed by the uni-system, see Figure 1). As shown in Figure 2a, the hsa-mir-9-5p demonstrated qPCR efficiency between 90 ~ 110% (N=3, STD <= 0.41), which is considered acceptable (Robledo et al 2014). To evaluate the qPCR efficiency of specific-FR-system, the oligonucleotides hsa-let-7b-5p, member of the let-7 miRNA family, was selected as a template for the miRNA serial titration assay.…”