Evaluation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in Pyrus pyrifolia using different tissue samples and seasonal conditions
Plant Physiology and Fruit Chemistry Division, NARO Institute of Fruit Tree Science, Tsukuba, Ibaraki, Japan; Biotechnology Research and Development Centre, Ebonyi State University, Abakaliki, Ebonyi State, Nigeria; Graduate School of Life and Environment
We have evaluated suitable reference genes for real time (RT)-quantitative PCR (qPCR) analysis in Japanese pear (Pyrus pyrifolia). We tested most frequently used genes in the literature such as β-Tubulin, Histone H3, Actin, Elongation factor-1α, Glyceraldehyde-3-phosphate dehydrogenase, together with newly added genes Annexin, SAND and TIP41. A total of 17 primer combinations for these eight genes were evaluated using cDNAs synthesized from 16 tissue samples from four groups, namely: flower bud, flower organ, fruit flesh and fruit skin. Gene expression stabilities were analyzed using geNorm and NormFinder software packages or by ΔCt method. geNorm analysis indicated three best performing genes as being sufficient for reliable normalization of RT-qPCR data. Suitable reference genes were different among sample groups, suggesting the importance of validation of gene expression stability of reference genes in the samples of interest. Ranking of stability was basically similar between geNorm and NormFinder, suggesting usefulness of these programs based on different algorithms. ΔCt method suggested somewhat different results in some groups such as flower organ or fruit skin; though the overall results were in good correlation with geNorm or NormFinder. Gene expression of two cold-inducible genes PpCBF2 and PpCBF4 were quantified using the three most and the three least stable reference genes suggested by geNorm. Although normalized quantities were different between them, the relative quantities within a group of samples were similar even when the least stable reference genes were used. Our data suggested that using the geometric mean value of three reference genes for normalization is quite a reliable approach to evaluating gene expression by RT-qPCR. We propose that the initial evaluation of gene expression stability by ΔCt method, and subsequent evaluation by geNorm or NormFinder for limited number of superior gene candidates will be a practical way of finding out reliable reference genes. © 2014 Imai et al.
actin; annexin; beta tubulin; complementary DNA; elongation factor 1alpha; glyceraldehyde 3 phosphate dehydrogenase; histone H3; membrane protein; protein SAND; protein TIP41; unclassified drug; 3' untranslated region; animal cell; animal tissue; article; controlled study; gene amplification; gene expression; gene locus; gene targeting; genetic analysis; genetic stability; molecular cloning; nonhuman; nucleotide sequence; plant development; plant gene; plant genetics; plant structures; PpCBF2 gene; PpCBF4 gene; Pyrus; Pyrus pyrifolia; quantitative analysis; real time polymerase chain reaction; seasonal variation; 3' Untranslated Regions; Computational Biology; Gene Expression Profiling; Gene Expression Regulation, Plant; Genes, Plant; Open Reading Frames; Organ Specificity; Pyrus; Real-Time Polymerase Chain Reaction; RNA Stability; Seasons
JSPS, Japan Society for the Promotion of Science