Selection and Optimization of Reference Genes for MicroRNA Expression Normalization by qRT-PCR in Chinese Cedar (Cryptomeria fortunei) under Multiple Stresses.

Yingting Zhang,Hailiang Hu,Junjie Yang,Jiebing Cui,Jin Xu,Jinyu Xue,Lijuan Zhu

doi:10.3390/ijms22147246

Yingting Zhang, Hailiang Hu + Show 5 more

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https://doi.org/10.3390/ijms22147246

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Abstract

MicroRNA (miRNA) expression analysis is very important for investigating its functions. To date, no research on reference genes (RGs) for miRNAs in gymnosperms, including Cryptomeria fortunei, has been reported. Here, ten miRNAs (i.e., pab-miR159a, cln-miR162, cas-miR166d, pab-miR395b, ppt-miR894, cln-miR6725, novel1, novel6, novel14 and novel16) and three common RGs (U6, 5S and 18S) were selected as candidate RGs. qRT-PCR was used to analyse their expressions in C. fortunei under various experimental conditions, including multiple stresses (cold, heat, drought, salt, abscisic acid and gibberellin) and in various tissues (roots, stems, tender needles, cones and seeds). Four algorithms (delta Ct, geNorm, NormFinder and BestKeeper) were employed to assess the stability of candidate RG expression; the geometric mean and RefFinder program were used to comprehensively evaluate RG stability. According to the results, novel16, cln-miR6725, novel1 and U6 were the most stable RGs for studying C. fortunei miRNA expression. In addition, the expression of three target miRNAs (aly-miR164c-5p, aly-miR168a-5p and smo-miR396) was examined to verify that the selected RGs are suitable for miRNA expression normalisation. This study may aid further investigations of miRNA expression/function in the response of C. fortunei to abiotic stress and provides an important basis for the standardisation of miRNA expression in other gymnosperm species.

Highlights

The expression of three target miRNAs, i.e., aly-miR164c-5p, alymiR168a-5p and smo-miR396, was used to verify that the selected reference genes (RGs) are suitable for gene-expression normalisation in different tissues or under the selected treatment. These results identified appropriate RGs that can be used to normalise the expression of miRNAs in C. fortunei, providing a basis for normalising miRNA expression in other coniferous species
Melting curves and 2.5% (w/v) agarose gel electrophoresis showed that each pair of primers used to amplify a candidate RG produced a single PCR-specific product of the desired size (Figure 1), indicating that all primer pairs used for RG selection had good primer specificity
Sci. 2021, 22, 7246 curves and 2.5% (w/v) agarose gel electrophoresis showed that each pair of primers used to amplify a candidate RG produced a single PCR-specific product of the desired size (Figure 1), indicating that all primer pairs used for RG selection had good primer specifor PCR

Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Chinese cedar (Cryptomeria fortunei) belongs to Cupressaceae. Because of its rapid growth, straight trunk and good wood texture, C. fortunei has become one of the main fast-growing timber afforestation species in subtropical high-elevation areas in China and has broad application prospects. C. fortunei usually grows in warm and humid climates, and its growth is often affected by adverse environmental conditions, such as low temperature, acid/aluminium and other stresses [1,2,3]. Molecular biology studies of Cryptomeria have largely focused on analyses of functional genes to reveal the growth and development mechanisms of these trees, whereas few studies have examined microRNAs (miRNAs) [4,5,6]

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