alidation of reference genes for quantitative expression analysis by qPCR in various tissues of date mussel (Lithophagalithophaga)

Abstract

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