Validation of reference genes for quantitative expression analysis by qPCR in various tissues of date mussel (Lithophaga lithophaga)

Abstract

qPCR is a very popular method for identifying nucleotide sequences as a result of its sensitivity and relatively low cost andtechnical simplicity. Normalization is one of the most important steps in analyzing qPCR data and the use of reference genes is themost common normalization strategy. In the present study five commonly used reference genes (18S, 28S, ef1a, β-act, and α-tub) wereevaluated for their stability in the mantle, gill, foot, and pallial gland of date mussel (L. lithophaga) in different seasons. Four differentsoftware packages were used for evaluation: geNorm, NormFinder, BestKeeper, and RefFinder. Although these programs containdifferent algorithms and analytical procedures, their ranking of the candidate reference genes was similar. Of the five selected referencegenes 18S, 28S, and ef1a were determined as the three most stable in different tissues and seasons. A-tub was evaluated as the least stablereference gene and therefore inappropriate for normalization of quantitative gene expression data. The results will help improve theaccuracy of gene expression analysis in samples of date mussel and at the same time provide guidance for selection of reference genesin future qPCR studies in the species.